Matrix metalloproteinases

ABSTRACT

The present invention provides genes encoding novel matrix metalloproteinases termed MMP; constructs and recombinant host cells incorporating the genes; the MMP polypeptides encoded by the genes; antibodies to the MMP polypeptides; and methods of making and using all of the foregoing.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority of application Ser. No.60/206,119, filed May 22, 2000 which is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the fields of genetics andcellular and molecular biology. More particularly, the invention relatesto novel matrix metalloproteinases.

BACKGROUND OF THE INVENTION

Matrix metalloproteinases (MMPs; matrixins) comprise a family ofstructurally related Zn-containing proteases that degrade allmacromolecules present in the extracellular matrix (ECM). Each of theknown MMPs can be divided up into a variable number of well-conserveddomains. All contain a pro-peptide which is involved in suppressing theactivity of the pro-enzyme form of the molecule, and a HEXXH sequencemotif that has been shown by X-ray crystallography to form part of themetal-binding site (Nagase et al. (1999) J. Biol. Chem., 274, 31,21491-21494). In addition, fibronectin-, hemopexin-, or vitronectin-likedomains and/or a membrane “anchor” domain may also be present.

Today, the MMP family includes more than 15 members (Table 1).

TABLE 1 Characteristics of known human MMPs MMP Type Substrate MMP-1Collagenase collagen I, II, III, VII, X gelatins MMP-2 Gelatinase Bcollagens IV, V, VII, XI, fibronectin, elastin MMP-3 Stromelysin 1proteoglycans, gelatins, fibronectin, collagens II, IV, IX MMP-7Matrilysin Proteoglycans, gelatins, collagen TV, elastin MMP-8Neutrophil collagens I, II, III collagenase MMP-9 Gelatinase B gelatins,collagen IV, V, proteoglycan, elastin MMP-10 Stromelysin 2 proteoglycan,fibronectin, laminin MMP-12 Macrophage proteoglycans, elastase elastaseMMP-13 Collagenase 3 collagens I, II, III, IV

MMPs are believed to play a critical role in many physiological andpathological processes. The breakdown of ECM by MMPs is essential forprocesses including embryonic development, morphogenesis, reproduction,and tissue repair and remodeling. Other physiological processes whichinvolve MMPs include tumor growth, tumor invasion, Sjögren's syndrome,periodontal diseases, arthritis, cardiomyopathy, renal failure,atherosclerosis, insulin resistance, adipogenesis, retinalneovascularization, wound healing, and neurodegenerative diseasesincluding, for example, Alzheimer's disease, multiple sclerosis,Parkinson's disease, and motoneuron disease. Identification of thefunctions of additional genes of the MMP family would be invaluable tothose of skill in the art seeking to understand the genetic basis forthese processes, as well as identifying compounds that modulate theactivity of such genes useful in methods for treating the pathologies.

Tissue inhibitors of metalloproteinases (TIMPs) are a group of closelyrelated secreted proteins that limit MMP activity. To date, four TIMPshave been characterized; TIMP1, TIMP2, TIMP3, and TIMP4, respectively(Gomez et al. (1997) Eur. J. Cell. Biol., 74, 111-122). Severalinvestigators have studied effects of MMP inhibition by using cellsover-expressing TIMPs. The balance between MMPs and TIMPs seems to playan important role in matrix turnover in several organ systems.

The past few years have witnessed several advances in the understandingof the pathophysiology of coronary atherosclerosis. The earliestatherosclerotic lesion, named the fatty streak, represents a dynamicbalance of the entry and exit of lipoprotein as well as the developmentof extracellular matrix. A decrease in lipoprotein entry will probablyresult in a predominance of lipoprotein exit and final scarring.However, an increase of lipoprotein entry can predominate over theefflux and scarring, resulting in vulnerable lipid-rich plaques that areprone to disruption (Falk et al., (1995), Circulation, 92:657-671;Fuster et al., (1999), Lancet, 353:SII: 5-9).

It is evident from many studies that MMPs, as a family, are importantregulators of atherosclerotic plaque growth (Newby et al., (1994), BasicRes. Cardiol. 89 [Suppl. 1] 59-70). However, the roles of the individualMMPs are so far largely unknown. Several MMPs are expressed in thediseased blood vessel, i.e. in smooth muscle cells and in macrophages.MMPs likely regulate both the degradation of extracellular matrix andinfluence the proliferation rate of smooth muscle cells. Severalinflammatory cytokines and growth factors increase the expression ofMMPs in cell cultures, e.g. interleukin-1, platelet-derived growthfactor (PDGF) and tumor necrosis factor-α (TNF-α).

It has been demonstrated in several animal models that inhibition ofMMPs (type 1 and 2 among others) decreases smooth muscle proliferationin response to vascular damage. Moreover, MMPs seem to enhance smoothmuscle cell migration. These two physiological processes are hallmarksof the neointimal thickening that characterizes atherosclerosis.Accordingly, MMP inhibitors may delay or prevent spontaneousatherogenesis as well as restenosis. MMPs and/or TIMPs may be especiallyuseful for patients at risk for atherosclerosis, dyslipidemia, end-stagerenal failure, or patients who have undergone Percutaneous TransluminalCoronary Angioplasty Procedure (PTCA).

A large number of studies support a role of MMPs in intima mediafunction. For example, over-expression of TIMP2 inhibits vascular smoothmuscle cell proliferation and chemotaxis in vitro (Baker et al., (1998),J. Clin. Invest., 101:1478-1487; Cheng et al., (1998), Circulation,98:2195-2201). In addition, it has been shown that MMPs are linked tothe proliferation and outgrowth of vascular smooth muscle cells fromexplants of rabbit aorta in vitro. The proliferation and outgrowth ofvascular smooth muscle cells from rabbit aorta was blocked byexperimental inhibitors (Ro 31-4724 and Ro 31-7467) (Newby et al.,(1994). Batimastat (BB94), a synthetic MMP inhibitor, can reduce smoothmuscle cell proliferation in vitro as well as inhibit neointimalformation after balloon injury to the rat carotid artery (Zempo et al.,(1996), Artherioscler. Thromb. Vasc. Biol., 16:28-33). Localoverexpression of TIMP1 has been shown to inhibit intimal hyperplasia inrats (Forough et al., (1996), Circ. Res., 79:812-820). After in vitroincubation with MMP-3, -7, or -12, the ability of HDL(3) to induce thehigh affinity component of cholesterol efflux from the macrophage foamcells was strongly reduced (Lindstedt et al., (1999), J. Biol. Chem.,274:22627-22634).

Angiogenesis, also known as neovascularization or new vessel growth, ispart of the normal wound healing machinery and can occur as a reactionto tissue hypoxia. Various tumors are also known to triggerangiogenesis, leading to tumor growth. In normal adult tissue, there isa balance between angiogenic and anti-angiogenic factors and, as aresult, few new vessels are formed. However, if the balance betweenangiogenic and anti-angiogenic factors is disturbed, a complex cascadeof events can be triggered that eventually leads to the formation of newblood vessels.

Diabetic retinopathy is the leading cause of blindness for the majorityof Americans. Microvascular damage from diabetes leads tomicroaneurysms, hemorrhage, exudates, and cotton-wool spots. Furtherprogression of disease leads to neovascularization. Growth of new bloodvessels can cause severe hemorrhage, scarring, and permanent visual loss(for a review, see Frank et al, (1996), South. Med. J., 89:463-470;Jampol & Goldbaum, (1980), Surv. Ophthalmol. 25:1-14). Variousrandomized, prospective studies have clearly shown benefit from lasertherapy at specific stages of progression of retinopathy.

AMD with rapid progression (wet AMD) is another common cause ofblindness in the developed world. Presently the underlying etiology ofAMD is unknown but a slow deterioration of the retinal pigmentepithelium, leading to the death of macular photoreceptors, is believedto be an important factor. The wet form of AMD often leads to a completeloss of central vision within a few years. AMD usually debuts in the dryform and may subsequently change into the wet form. AMD with rapidprogression is characterized by choroidal new vessel formation (CNV).The new vessels tend to leak and may rupture. The resulting macularedema, bleeding, fibrinous deposits, and scar formation are reasons forthe rapid deterioration of vision in this form of AMD.

Sprouting is a key step in CNV formation. If sprouting can be inhibited,no new leaky vessels will form. MMPs are essential to create space forthe new sprouts. Because this step is downstream in the angiogenesisprocess, an MMP inhibitor can work to limit sprouting even if theearlier events are slightly different from those described above. Thelocalization of MMP-2 and MMP-9 to the areas of new vessel formation andto the enveloping Bruch's-like membrane, respectively, suggests thatMMP-2 and MMP-9 may be cooperatively involved in the progressive growthof choroidal neovascular membranes (Steen et al. (1998), Invest.Ophthalmol. Vis. Sci., 39:2194-2200). In normal individuals MMP-9activity is not detected in the eye; however, it has been demonstratedthat MMP-9 activity is detected in more than 80% of patients with“active” proliferative retinopathy (Kosano et al., (1999), Life Sci.,64:2307-2315).

MMP inhibitors present an attractive opening for prophylacticpharmacotherapy of ocular blood vessel proliferation in diabetes andAMD. Moreover, it may be possible to combine MMP inhibitors withphotodynamic therapy. It is possible that inhibitors of MMPs couldprevent recurrence of CNV and, thus, improve long-term efficacy.Patients are likely to accept certain side effects in order to preservetheir vision, as most are aware that the disease will rapidly lead toblindness. Topical treatment is advantageous from a pharmacovigilancepoint-of-view.

MMPs are also involved in the bioacticvation of cytokines, includingtumor necrosis factor-alpha (TNF-α). Evidence suggests that TNF-α is akey mediator of insulin resistance in adipocytes and skeletal muscle.Inhibition of MMPs may decrease the formation rate of TNF-α and,accordingly, be of therapeutic significance in type-II diabetes. MMPsand/or TIMPS may be useful for patients with Type II diabetes or forobese patients with insulin resistance.

It has been suggested that TNF-α is an inducer of insulin resistance intype II diabetes. TNF-α is synthesized as a membrane-bound precursorthat is proteolytically processed to an active form by a matrixmetalloproteinase (MMP)-like enzyme. It has been shown that subcutaneousadministration of KB-R7785 (a non-specific MMP inhibitor) to KKAy mice,which show insulin resistance and hyperglycemia for 4 weeks, resulted ina significant decrease in plasma glucose levels after 3 weeks ofadministration. In the same study it was also demonstrated thatadministration of pioglitazone significantly decreased plasma glucoselevels. Interestingly, KB-R7785, but not pioglitazone, alsosignificantly decreased plasma insulin levels in the animals. It hasalso been shown that the lipopolysaccharide-induction of TNF-α in plasmacan be inhibited in KKAy mice by KB-R7785. These results suggest thatMNMP inhibitors may exert an anti-diabetic effect by amelioratinginsulin sensitivity through the inhibition of TNF-α production.

Nephropathy in patients with type I and II diabetes mellitus is arapidly increasing problem worldwide. Diabetic patients account fornearly half of all patients on hemodialysis. Microalbuminuria isdiagnosed when the urinary albumin excretion rate is greater than 20 butless than 200 micrograms/min and the prevalence of microalbuminuriaamong diabetic patients is 15-20% (Deckert et al., (1992), DiabetesCare, 15:1181-1191).

MMP inhibitors (non-selective) have been found to decrease theproliferation rate of cultured rat mesangial cells without affectingcell viability. Therefore, MMP inhibitors may offer a new therapeuticapproach for treatment of mesangial cell-derived forms ofglomerulonephritis and prevent basal membrane thickening in diabetes.MMPs and/or TIMPS may be useful for diabetic patients with early signsof glomerulopathy, or for patients with microalbuminuria.

Progressive expansion of the mesangial matrix, and thickening of theglomerular and tubular basement membranes are hallmarks of human andexperimental diabetic nephropathy (Philips et al. (1999), Kidney BloodPress. Res. 22:81-97; Young et al., (1995) Kidney Int., 47:935-944).These lesions eventually lead to glomerular fibrosis, a centralpathological feature in many human acute and chronic kidney diseases,which progressively destroys the renal filtration unit, and may finallycause renal failure. It has been demonstrated that mesangial matrixexpansion is strongly related to the clinical manifestation of diabeticnephropathy. Diabetic nephropathy is effected both directly andindirectly by the alteration of cytokine generation. Data from studieson several animal species suggest that proliferation of mesangial cellsis an important feature of diabetic glomerulopathy. Harendza et al.,(Nephrol. Dial. Transplant 12:2537-2541, (1997)) have demonstrated thatthe expression of MMP2 is enhanced in experimental proliferativeglomerulopathy in the rat. Inhibition of MMP2 by Ro 31-9790 inhibitedthe proliferation rate of cultured rat mesangial cells in aconcentration-dependent and at least partially reversible manner withoutaffecting cell viability (Steinmann-Niggli K, et al., (1997), J. Am.Soc. Nephrol. 8:395-405). Moreover, Ebihara et al., ((1998) Am. J.Kidney Dis. 32:544-550) have reported that increased MMP9 concentrationsin plasma preceded the occurrence of microalbuminuria in diabeticpatients.

Thus, a need exists for new members of the MMP family of proteases.

SUMMARY OF THE INVENTION

The present invention addresses the need identified above by providingDNA sequences of genes encoding heretofore unknown members of the MMPfamily of proteases, bearing sequence homology and functional homologyto MMPs; constructs and recombinant host cells incorporating the genes;the MMP polypeptides encoded by the genes; antibodies to thepolypeptides; kits employing the polynucleotides and polypeptides, andmethods of making and using all of the foregoing. Exemplary diseases andconditions amenable to treatment based on the present invention include,but are not limited to metabolic diseases and disorders (e.g., type 2diabetes, obesity, cardiovascular, dyslipidemias, adipogenesis,retinopathies, neuropathies, nephropathies etc.), proliferative diseasesand cancers (e.g., different cancers such as breast, colon, lung, etc.,tumor growth, tumor invasion, and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc.), hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.), CNS disorders (e.g., degenerative disorders such as Parkinson's,Alzheimer's, etc.), inflammatory conditions (e.g., Chron's disease,arthritis), diseases related to cell differentiation and homeostasis,cardiomyopathy, atherosclerosis, thromboembolic diseases, Sjögren'ssyndrome, renal failure, periodontal diseases, retinalneovascularization, wound healing, and neurodegenerative diseasesincluding, for example, Alzheimer's disease, multiple sclerosis,Parkinson's disease, and motoneuron disease, among others.

In one embodiment, the invention provides purified and isolated MMPpolypeptides comprising the amino acid sequence set forth in SEQ ID NOS:4-6, or a fragment thereof comprising an epitope specific to the MMPpolypeptide. Preferred embodiments comprise purified and isolatedpolypeptides comprising the complete amino acid sequences set forth inany of SEQ ID NOS: 4-6, found in Table 5 below. These amino acidsequences were deduced from the polynucleotide sequences encodingMMP-(SEQ ID NOS: 1-3 found in Table 5 below).

In another preferred embodiment, the invention provides a purified andisolated polypeptide comprising at least one conserved MMP domain.

In another embodiment, the invention provides purified and isolatedpolynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, orcombinations thereof, whether single- or double-stranded) that comprisea nucleotide sequence encoding the amino acid sequence of thepolypeptides of the invention. Such polynucleotides are useful forrecombinantly expressing the protease and also for detecting expressionof the protease in cells (e.g., using Northern hybridization and in situhybridization assays. Such polynucleotides also are useful in the designof antisense and other molecules for the suppression of the expressionof MMPs in a cultured cell, a tissue, or an animal; for therapeuticpurposes; or to provide a model for diseases or conditions characterizedby aberrant MMP expression. Specifically excluded from the definition ofpolynucleotides of the invention are entire isolated, non-recombinantnative chromosomes of host cells. Preferred polynucleotides have thesequences set forth in SEQ ID NOS: 1-3, which correspond to a naturallyoccurring MMP-sequences.

The invention also provides a purified and isolated polynucleotidecomprising a nucleotide sequence that encodes a mammalian gene product,wherein the polynucleotide hybridizes to a polynucleotide having thesequences set forth in SEQ ID NOS: 1-3 or the non-coding strandcomplementary thereto, under the following hybridization conditions:

(a) hybridization for 16 hours at 42° C. in a hybridization solutioncomprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate; and

(b) washing 2 times for 30 minutes each at 60° C. in a wash solutioncomprising 0.1% SSC, 1% SDS. Polynucleotides that encode a human allelicvariant are highly preferred.

In a related embodiment, the invention provides vectors comprising apolynucleotide of the invention. Such vectors are useful, e.g., foramplifying the polynucleotides in host cells to create useful quantitiesthereof. In preferred embodiments, the vector is an expression vectorwherein the polynucleotide of the invention is operatively linked to apolynucleotide comprising an expression control sequence. Such vectorsare useful for recombinant production of polypeptides of the invention.

In another related embodiment, the invention provides host cells thatare transformed or transfected (stably or transiently) withpolynucleotides of the invention or vectors of the invention. Such hostcells are useful for amplifying the polynucleotides and also forexpressing the MMP polypeptides or fragments thereof encoded by thepolynucleotides.

In still another related embodiment, the invention provides a method forproducing MMP polypeptides (or fragments thereof) comprising the stepsof growing a host cell of the invention in a nutrient medium andisolating the polypeptide or variant thereof from the cell or themedium.

In still another embodiment, the invention provides an antibody that isspecific for the MMP of the invention. Antibody specificity is describedin greater detail below. However, it should be emphasized thatantibodies that can be generated from polypeptides that have previouslybeen described in the literature and that are capable of fortuitouslycross-reacting with MMP (e.g., due to the fortuitous existence of asimilar epitope in both polypeptides) are considered “cross-reactive”antibodies. Such cross-reactive antibodies are not antibodies that are“specific” for MMP.

In one preferred variation, the invention provides monoclonalantibodies. Hybridomas that produce such antibodies also are intended asaspects of the invention. In yet another variation, the inventionprovides a humanized antibody. Humanized antibodies are useful for invivo therapeutic indications.

In another variation, the invention provides a cell-free compositioncomprising polyclonal antibodies, wherein at least one of the antibodiesis an antibody of the invention specific for MMP. Antisera isolated froman animal is an exemplary composition, as is a composition comprising anantibody fraction of an antisera that has been resuspended in water orin another diluent, excipient, or carrier.

In still another related embodiment, the invention provides ananti-idiotypic antibody specific for an antibody that is specific forMMP.

In still another embodiment, the invention provides a polypeptidecomprising a fragment of an MMP-specific antibody, wherein the fragmentand the polypeptide bind to the MMP. By way of non-limiting example, theinvention provides polypeptides that are single chain antibodies andCDR-grafted antibodies.

Also within the scope of the invention are compositions comprisingpolypeptides, polynucleotides, or antibodies of the invention that havebeen formulated with, e.g., a pharmaceutically acceptable carrier.

The invention also provides methods of using antibodies of theinvention. For example, the invention provides a method for modulatingsubstrate binding of a MMP comprising the step of contacting the MMPwith an antibody specific for the MMP, under conditions wherein theantibody binds the MMP.

MMPs may be expressed in various tissues, and an expression profile ofsuch tissues provides additional uses for the invention. For example, ifMMP is expressed in the brain, it would provide an indication thataberrant MMP activity may correlate with one or more neurologicaldisorders. The invention thus also provides a method for treating aneurological disorder comprising the step of administering to a mammalin need of such treatment an amount of an antibody-like polypeptide ofthe invention that is sufficient to modulate substrate binding to a MMPin neurons of the mammal. MMP may also be expressed in other tissues,including but not limited to pancreas (and particularly pancreatic islettissue), pituitary, skeletal muscle, adipose tissue, liver, and thyroid.

The invention also provides assays to identify compounds that bind aMMP. One such assay comprises the steps of: (a) contacting a compositioncomprising a MMP with a compound suspected of binding MMP; and (b)measuring binding between the compound and MMP. In one variation, thecomposition comprises a cell expressing MMP.

The invention also provides a method for identifying a modulator ofbinding between a MMP and a MMP binding partner, comprising the stepsof: (a) contacting a MMP binding partner and a composition comprising aMMP in the presence and in the absence of a putative modulator compound;(b) detecting binding between the binding partner and the MMP; and (c)identifying a putative modulator compound or a modulator compound inview of decreased or increased binding between the binding partner andthe MMP in the presence of the putative modulator, as compared tobinding in the absence of the putative modulator.

MMP binding partners that stimulate MMP activity are useful asactivators in disease states or conditions characterized by insufficientMMP activity. MMP binding partners that block MMP-mediated proteolysisare useful as MMP repressors to treat disease states or conditionscharacterized by excessive MMP-mediated proteolysis. In addition MMPproteolysis modulators in general, as well as MMP polynucleotides andpolypeptides, are useful in diagnostic assays for such diseases orconditions.

In another aspect, the invention provides methods for treating a diseaseor abnormal condition by administering to a patient in need of suchtreatment a substance that modulates the activity or expression of MMP.Preferably, the disease is selected from the group consisting of tumorgrowth, tumor invasion, Sjögren's syndrome, periodontal diseases,arthritis, cardiomyopathy, renal failure, atherosclerosis, insulinresistance, adipogenesis, retinal neovascularization, wound healing, andneurodegenerative diseases including, for example, Alzheimer's disease,multiple sclerosis, Parkinson's disease, and motoneuron disease.

In another aspect, the invention features methods for detection of apolypeptide in a sample as a diagnostic tool for diseases or disorders,wherein the method comprises the steps of: (a) contacting the samplewith a nucleic acid probe which hybridizes under hybridization assayconditions to a nucleic acid target region of a MMP polypeptide, saidprobe comprising the nucleic acid sequence encoding the polypeptide,fragments thereof, and the complements of the sequences and fragments;and (b) detecting the presence or amount of the probe:target regionhybrid as an indication of the disease.

The diseases for which detection of genes in a sample could bediagnostic include diseases in which nucleic acid (DNA and/or RNA) isamplified in comparison to normal cells. The diseases that could bediagnosed by detection of nucleic acid in a sample preferably includecancers. The test samples suitable for nucleic acid probing methods ofthe present invention include, for example, cells or nucleic acidextracts of cells, or biological fluids. The samples used in theabove-described methods will vary based on the assay format, thedetection method and the nature of the tissues, cells or extracts to beassayed. Methods for preparing nucleic acid extracts of cells are wellknown in the art and can be readily adapted in order to obtain a samplethat is compatible with the method utilized.

Additional features and variations of the invention will be apparent tothose skilled in the art from the entirety of this application,including the detailed description, and all such features are intendedas aspects of the invention. Likewise, features of the inventiondescribed herein can be re-combined into additional embodiments thatalso are intended as aspects of the invention, irrespective of whetherthe combination of features is specifically mentioned above as an aspector embodiment of the invention. Also, only such limitations that aredescribed herein as critical to the invention should be viewed as such;variations of the invention lacking limitations that have not beendescribed herein as critical are intended as aspects of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides purified and isolated polynucleotides(e.g., DNA sequences and RNA transcripts, both sense and complementaryantisense strands, both single- and double-stranded, including splicevariants thereof) encoding a matrix metalloproteinase referred to hereinas MMP. DNA polynucleotides of the invention include genomic DNA, cDNA,and DNA that has been chemically synthesized in whole or in part.

Definitions

Various definitions are made throughout this document. Most words havethe meaning that would be attributed to those words by one skilled inthe art. Words specifically defined either below or elsewhere in thisdocument have the meaning provided in the context of the presentinvention as a whole and as are typically understood by those skilled inthe art.

“Synthesized” as used herein and understood in the art, refers topolynucleotides produced by purely chemical, as opposed to enzymatic,methods. “Wholly” synthesized DNA sequences are therefore producedentirely by chemical means, and “partially” synthesized DNAs embracethose wherein only portions of the resulting DNA were produced bychemical means.

By the term “region” is meant a physically contiguous portion of theprimary structure of a biomolecule. In the case of proteins, a region isdefined by a contiguous portion of the amino acid sequence of thatprotein.

The term “domain” is herein defined as referring to a structural part ofa biomolecule that contributes to a known or suspected function of thebiomolecule. Domains may be co-extensive with regions or portionsthereof; domains may also incorporate a portion of a biomolecule that isdistinct from a particular region, in addition to all or part of thatregion.

As used herein, the terms “ligand” and “binding partner” are usedinterchangeably and refer to compounds that bind to proteases such asMMP or portions of such proteases.

Unless indicated otherwise, as used herein the abbreviation in lowercase (mmp) refers to a gene, cDNA, RNA or nucleic acid sequence whilethe upper case version (MMP) refers to a protein, polypeptide, peptide,oligopeptide, or amino acid sequence.

As used herein, the term “activity” refers to a variety of measurableindicia suggesting or revealing binding, either direct or indirect;affecting a response, i.e. having a measurable affect in response tosome exposure or stimulus, including, for example, the affinity of acompound for directly binding a polypeptide or polynucleotide of theinvention, or, for example, measurement of amounts of upstream ordownstream proteins or other similar functions after some stimulus orevent.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab, Fab′, F(ab)2, and other fragments thereof.Complete, intact antibodies include monoclonal antibodies such as murinemonoclonal antibodies, chimeric antibodies and humanized antibodies.

As used herein, the term “binding” means the physical or chemicalinteraction between two proteins or compounds or associated proteins orcompounds or combinations thereof. Binding includes ionic, non-ionic,Hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Thephysical interaction, the binding, can be either direct or indirect,indirect being through or due to the effects of another protein orcompound. Direct binding refers to interactions that do not take placethrough or due to the effect of another protein or compound but insteadare without other substantial chemical intermediates. Binding may bedetected in many different manners. As a non-limiting example, thephysical binding interaction between a MMP of the invention and acompound can be detected using a labeled compound. Alternatively,functional evidence of binding can be detected using, for example, acell transfected with and expressing an MMP of the invention. Binding ofthe transfected cell to a ligand of the MMP that was transfected intothe cell provides functional evidence of binding. Other methods ofdetecting binding are well known to those of skill in the art.

As used herein, the term “compound” means any identifiable chemical ormolecule, including, but not limited to, small molecule, peptide,protein, sugar, nucleotide, or nucleic acid, and such compound can benatural or synthetic.

As used herein, the term “complementary” refers to Watson-Crickbasepairing between nucleotide units of a nucleic acid molecule.

As used herein, the term “contacting” means bringing together, eitherdirectly or indirectly, a compound into physical proximity to apolypeptide or polynucleotide of the invention. The polypeptide orpolynucleotide can be in any number of buffers, salts, solutions etc.Contacting includes, for example, placing the compound into a beaker,microtiter plate, cell culture flask, or a microarray, such as a genechip, or the like, which contains the nucleic acid molecule, orpolypeptide encoding the MMP or fragment thereof.

As used herein, the phrase “homologous nucleotide sequence,” or“homologous amino acid sequence,” or variations thereof, refers tosequences characterized by a homology, at the nucleotide level or aminoacid level, of at least the specified percentage. Homologous nucleotidesequences include those sequences coding for isoforms of proteins. Suchisoforms can be expressed in different tissues of the same organism as aresult of, for example, alternative splicing of RNA. Alternatively,isoforms can be encoded by different genes. Homologous nucleotidesequences include nucleotide sequences encoding for a protein of aspecies other than humans, including, but not limited to, mammals.Homologous nucleotide sequences also include, but are not limited to,naturally occurring allelic variations and mutations of the nucleotidesequences set forth herein. A homologous nucleotide sequence does not,however, include the nucleotide sequence encoding other known MMPs.Homologous amino acid sequences include those amino acid sequences whichcontain conservative amino acid substitutions and which polypeptideshave the same binding and/or activity. A homologous amino acid sequencedoes not, however, include the amino acid sequence encoding other knownMMPs. Percent homology can be determined by, for example, the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, Madison Wis.), usingthe default settings, which uses the algorithm of Smith and Waterman(Adv. Appl. Math., 1981, 2, 482-489, which is incorporated herein byreference in its entirety).

As used herein, the term “isolated” nucleic acid molecule refers to anucleic acid molecule (DNA or RNA) that has been removed from its nativeenvironment. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules.

As used herein, the terms “modulates” or “modifies” means an increase ordecrease in the amount, quality, or effect of a particular activity orprotein.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues which has a sufficient number of bases to be used ina polymerase chain reaction (PCR). This short sequence is based on (ordesigned from) a genomic or cDNA sequence and is used to amplify,confirm, or reveal the presence of an identical, similar orcomplementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a DNA sequence having at leastabout 10 nucleotides and as many as about 50 nucleotides, preferablyabout 15 to 30 nucleotides. They are chemically synthesized and may beused as probes.

As used herein, the term “probe” refers to nucleic acid sequences ofvariable length, preferably between at least about 10 and as many asabout 6,000 nucleotides, depending on use. They are used in thedetection of identical, similar, or complementary nucleic acidsequences. Longer length probes are usually obtained from a natural orrecombinant source, are highly specific and much slower to hybridizethan oligomers. They may be single- or double-stranded and carefullydesigned to have specificity in PCR, hybridization membrane-based, orELISA-like technologies.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops an abnormal condition.

The term “treating” refers to having a therapeutic effect and at leastpartially alleviating or abrogating an abnormal condition in theorganism.

The term “therapeutic effect” refers to the inhibition or activationfactors causing or contributing to the abnormal condition. A therapeuticeffect relieves to some extent one or more of the symptoms of theabnormal condition. In reference to the treatment of abnormalconditions, a therapeutic effect can refer to one or more of thefollowing: (a) an increase in the proliferation, growth, and/ordifferentiation of cells; (b) inhibition (i.e., slowing or stopping) ofcell death; (c) inhibition of degeneration; (d) relieving to some extentone or more of the symptoms associated with the abnormal condition; and(e) enhancing the function of the affected population of cells.Compounds demonstrating efficacy against abnormal conditions can beidentified as described herein.

The term “abnormal condition” refers to a function in the cells ortissues of an organism that deviates from their normal functions in thatorganism. An abnormal condition can relate to cell proliferation, celldifferentiation, cell signaling, or cell survival. An abnormal conditionmay also include obesity, diabetic complications such as retinaldegeneration, and irregularities in glucose uptake and metabolism, andfatty acid uptake and metabolism.

Abnormal cell proliferative conditions include cancers such as fibroticand mesangial disorders, abnormal angiogenesis and vasculogenesis, woundhealing, psoriasis, diabetes mellitus, and inflammation.

Abnormal differentiation conditions include, but are not limited to,neurodegenerative disorders, slow wound healing rates, and slow tissuegrafting healing rates. Abnormal cell signaling conditions include, butare not limited to, psychiatric disorders involving excessneurotransmitter activity.

Abnormal cell survival conditions may also relate to conditions in whichprogrammed cell death (apoptosis) pathways are activated or abrogated. Anumber of protein kinases are associated with the apoptosis pathways.Aberrations in the function of any one of the protein kinases could leadto cell immortality or premature cell death.

The term “aberration,” in conjunction with the function of an MMP,refers to an MMP that is over- or under-expressed in an organism,mutated such that its catalytic activity is lower or higher thanwild-type protease activity, mutated such that it can no longer interactwith a natural binding partner, or is no longer modified by anotherprotease.

The term “administering” relates to a method of incorporating a compoundinto cells or tissues of an organism. The abnormal condition can beprevented or treated when the cells or tissues of the organism existwithin the organism or outside of the organism. Cells existing outsidethe organism can be maintained or grown in cell culture dishes. Forcells harbored within the organism, many techniques exist in the art toadminister compounds, including (but not limited to) oral, parenteral,dermal, injection, and aerosol applications. For cells outside of theorganism, multiple techniques exist in the art to administer thecompounds, including (but not limited to) cell microinjectiontechniques, transformation techniques and carrier techniques.

The abnormal condition can also be prevented or treated by administeringa compound to a group of cells having an aberration in a signaltransduction pathway to an organism. The effect of administering acompound on organism function can then be monitored. The organism ispreferably a mouse, rat, rabbit, guinea pig or goat, more preferably amonkey or ape, and most preferably a human.

By “amplification” it is meant increased numbers of DNA or RNA in a cellcompared with normal cells. “Amplification” as it refers to RNA can bethe detectable presence of RNA in cells, since in some normal cellsthere is no basal expression of RNA. In other normal cells, a basallevel of expression exists, therefore in these cases amplification isthe detection of at least 1 to 2-fold, and preferably more, compared tothe basal level.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a probe, primer,or oligonucleotide will hybridize to its target sequence, but to noother sequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present in excess, at T_(m), 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g. 10 to 50 nucleotides) and at leastabout 60° C. for longer probes, primers or oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

The amino acid sequences are presented in the amino to carboxydirection, from left to right. The amino and carboxy groups are notpresented in the sequence. The nucleotide sequences are presented bysingle strand only, in the 5′ to 3′ direction, from left to right.Nucleotides and amino acids are represented in the manner recommended bythe IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids)by three letters code.

Polynucleotides

The present invention provides purified and isolated polynucleotides(e.g., DNA sequences and RNA transcripts, both sense and complementaryantisense strands, both single- and double-stranded, including splicevariants thereof) that encode unknown MMPs heretofore termed novel MMPs,or MMPs.

It is well known that MMPs are expressed in many different tissues,including the brain. Accordingly, the MMPs of the present invention maybe useful, inter alia, for treating and/or diagnosing mental disorders.Following the techniques described in Example 4, below, those skilled inthe art could readily ascertain if MMP is expressed in a particulartissue or region.

The invention provides purified and isolated polynucleotides (e.g.,cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whethersingle- or double-stranded) that comprise a nucleotide sequence encodingthe amino acid sequence of the polypeptides of the invention. Suchpolynucleotides are useful for recombinantly expressing the MMP and alsofor detecting expression of the MMP in cells (e.g., using Northernhybridization and in situ hybridization assays). Such polynucleotidesalso are useful in the design of antisense and other molecules for thesuppression of the expression of MMP in a cultured cell, a tissue, or ananimal; for therapeutic purposes; or to provide a model for diseases orconditions characterized by aberrant MMP expression. Specificallyexcluded from the definition of polynucleotides of the invention areentire isolated, non-recombinant native chromosomes of host cells. Apreferred polynucleotide has a sequence selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:3, which correspond to naturallyoccurring MMP sequences. It will be appreciated that numerous otherpolynucleotide sequences exist that also encode MMP having the sequenceselected from the group consisting of SEQ ID NO:4 to SEQ ID NO:6, due tothe well-known degeneracy of the universal genetic code.

The invention also provides a purified and isolated polynucleotidecomprising a nucleotide sequence that encodes a mammalian polypeptide,wherein the polynucleotide hybridizes to a polynucleotide having thesequence set forth in sequences selected from the group consisting ofSEQ ID NO:1 to SEQ ID NO:3, or the non-coding strand complementarythereto, under the following hybridization conditions:

(a) hybridization for 16 hours at 42° C. in a hybridization solutioncomprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate; and

(b) washing 2 times for 30 minutes each at 60° C. in a wash solutioncomprising 0.1% SSC, 1% SDS. Polynucleotides that encode a human allelicvariant are highly preferred.

The present invention relates to molecules which comprise the genesequences that encode the MMPs; constructs and recombinant host cellsincorporating the gene sequences; the novel MMP polypeptides encoded bythe gene sequences; antibodies to the polypeptides and homologs; kitsemploying the polynucleotides and polypeptides, and methods of makingand using all of the foregoing. In addition, the present inventionrelates to homologs of the gene sequences and of the polypeptides andmethods of making and using the same.

Genomic DNA of the invention comprises the protein-coding region for apolypeptide of the invention and is also intended to include allelicvariants thereof. It is widely understood that, for many genes, genomicDNA is transcribed into RNA transcripts that undergo one or moresplicing events wherein intron (i.e., non-coding regions) of thetranscripts are removed, or “spliced out.” RNA transcripts that can bespliced by alternative mechanisms, and therefore be subject to removalof different RNA sequences but still encode an MMP polypeptide, arereferred to in the art as splice variants which are embraced by theinvention. Splice variants comprehended by the invention therefore areencoded by the same original genomic DNA sequences but arise fromdistinct mRNA transcripts. Allelic variants are modified forms of awild-type gene sequence, the modification resulting from recombinationduring chromosomal segregation or exposure to conditions which give riseto genetic mutation. Allelic variants, like wild type genes, arenaturally occurring sequences (as opposed to non-naturally occurringvariants that arise from in vitro manipulation).

The invention also comprehends cDNA that is obtained through reversetranscription of an RNA polynucleotide encoding MMP (conventionallyfollowed by second strand synthesis of a complementary strand to providea double-stranded DNA).

Preferred DNA sequences encoding human MMP polypeptides are selectedfrom the group consisting of SEQ ID NO:1 to SEQ ID NO:3. A preferred DNAof the invention comprises a double stranded molecule along with thecomplementary molecule (the “non-coding strand” or “complement”) havinga sequence unambiguously deducible from the coding strand according toWatson-Crick base-pairing rules for DNA. Also preferred are otherpolynucleotides encoding the MMP polypeptide selected from the groupconsisting of SEQ ID NO:4 to SEQ ID NO:6, which differ in sequence fromthe polynucleotides selected from the group consisting of SEQ ID NO:1 toSEQ ID NO:3, by virtue of the well-known degeneracy of the universalnuclear genetic code.

The invention further embraces other species, preferably mammalian,homologs of the human MMP DNA. Species homologs, sometimes referred toas “orthologs,” in general, share at least 35%, at least 40%, at least45%, at least 50%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% homology with human DNA of the invention.Generally, percent sequence “homology” with respect to polynucleotidesof the invention may be calculated as the percentage of nucleotide basesin the candidate sequence that are identical to nucleotides in the MMPsequences set forth in sequences selected from the group consisting ofSEQ ID NO:1 to SEQ ID NO:3, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.

Polynucleotides of the invention permit identification and isolation ofpolynucleotides encoding related MMP polypeptides, such as human allelicvariants and species homologs, by well-known techniques includingSouthern and/or Northern hybridization, and polymerase chain reaction(PCR). Examples of related polynucleotides include human and non-humangenomic sequences, including allelic variants, as well aspolynucleotides encoding polypeptides homologous to MMP and structurallyrelated polypeptides sharing one or more biological, immunological,and/or physical properties of MMP. Non-human species genes encodingproteins homologous to MMP can also be identified by Southern and/or PCRanalysis and are useful in animal models for MMP disorders. Knowledge ofthe sequence of a human MMP DNA also makes possible through use ofSouthern hybridization or polymerase chain reaction (PCR) theidentification of genomic DNA sequences encoding expression controlregulatory sequences such as promoters, operators, enhancers,repressors, and the like. Polynucleotides of the invention are alsouseful in hybridization assays to detect the capacity of cells toexpress MMP. Polynucleotides of the invention may also provide a basisfor diagnostic methods useful for identifying a genetic alteration(s) inan MMP locus that underlies a disease state or states, which informationis useful both for diagnosis and for selection of therapeuticstrategies.

According to the present invention, the MMP nucleotide sequencesdisclosed herein may be used to identify homologs of the MMP, in otheranimals, including but not limited to humans and other mammals, andinvertebrates. Any of the nucleotide sequences disclosed herein, or anyportion thereof, can be used, for example, as probes to screen databasesor nucleic acid libraries, such as, for example, genomic or cDNAlibraries, to identify homologs, using screening procedures well knownto those skilled in the art. Accordingly, homologs having at least 50%,more preferably at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, more preferablyat least 95%, and most preferably at least 100% homology with MMPsequences can be identified.

The disclosure herein of full-length polynucleotides encoding MMPpolypeptides makes readily available to the worker of ordinary skill inthe art every possible fragment of the full-length polynucleotide.

In a preferred embodiment, the isolated nucleic acid comprises anucleotide sequence of SEQ ID NO: 2, and fragments thereof, that encodea polypeptide having a sequence of SEQ ID NO: 5, or fragments thereof.In a more preferred embodiment, the nucleotide is not SEQ ID NO:7 anddoes not encode a polypeptide with a sequence of SEQ ID NO:8.

As used in the present invention, fragments of MMP-encodingpolynucleotides comprise at least 10, and preferably at least 12, 14,16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotideencoding MMP. Preferably, fragment polynucleotides of the inventioncomprise sequences unique to the MMP-encoding polynucleotide sequence,and therefore hybridize under highly stringent or moderately stringentconditions only (i.e., “specifically”) to polynucleotides encoding MMP(or fragments thereof). Polynucleotide fragments of genomic sequences ofthe invention comprise not only sequences unique to the coding region,but also include fragments of the full-length sequence derived fromintrons, regulatory regions, and/or other non-translated sequences.Sequences unique to polynucleotides of the invention are recognizablethrough sequence comparison to other known polynucleotides, and can beidentified through use of alignment programs routinely utilized in theart, e.g., those made available in public sequence databases. Suchsequences also are recognizable from Southern hybridization analyses todetermine the number of fragments of genomic DNA to which apolynucleotide will hybridize. Polynucleotides of the invention can belabeled in a manner that permits their detection, including radioactive,fluorescent, and enzymatic labeling.

Fragment polynucleotides are particularly useful as probes for detectionof full-length or fragments of MMP polynucleotides. One or morepolynucleotides can be included in kits that are used to detect thepresence of a polynucleotide encoding MMP, or used to detect variationsin a polynucleotide sequence encoding MMP.

The invention also embraces DNAs encoding MMP polypeptides thathybridize under moderately stringent or high stringency conditions tothe non-coding strand, or complement, of the polynucleotides set forthin sequences selected from the group consisting of SEQ ID NO:1 to SEQ IDNO:3.

Exemplary highly stringent hybridization conditions are as follows:hybridization at 42° C. in a hybridization solution comprising 50%formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.It is understood in the art that conditions of equivalent stringency canbe achieved through variation of temperature and buffer, or saltconcentration as described Ausubel et al. (Eds.), Protocols in MolecularBiology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications inhybridization conditions can be empirically determined or preciselycalculated based on the length and the percentage of guanosine/cytosine(GC) base pairing of the probe. The hybridization conditions can becalculated as described in Sambrook, et al., (Eds.), Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold SpringHarbor, New York (1989), pp. 9.47 to 9.51.

With the knowledge of the nucleotide sequence information disclosed inthe present invention, one skilled in the art can identify and obtainnucleotide sequences which encode MMP from different sources (i.e.,different tissues or different organisms) through a variety of meanswell known to the skilled artisan and as disclosed by, for example,Sambrook et al., “Molecular cloning: a laboratory manual”, SecondEdition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989),which is incorporated herein by reference in its entirety.

For example, DNA that encodes MMP may be obtained by screening of mRNA,cDNA, or genomic DNA with oligonucleotide probes generated from the MMPgene sequence information provided herein. Probes may be labeled with adetectable group, such as a fluorescent group, a radioactive atom or achemiluminescent group in accordance with procedures known to theskilled artisan and used in conventional hybridization assays, asdescribed by, for example, Sambrook et al.

A nucleic acid molecule comprising any of the MMP nucleotide sequencesdescribed above can alternatively be synthesized by use of thepolymerase chain reaction (PCR) procedure, with the PCR oligonucleotideprimers produced from the nucleotide sequences provided herein. See U.S.Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 toMullis. The PCR reaction provides a method for selectively increasingthe concentration of a particular nucleic acid sequence even when thatsequence has not been previously purified and is present only in asingle copy in a particular sample. The method can be used to amplifyeither single- or double-stranded DNA. The essence of the methodinvolves the use of two oligonucleotide probes to serve as primers forthe template-dependent, polymerase mediated replication of a desirednucleic acid molecule.

A wide variety of alternative cloning and in vitro amplificationmethodologies are well known to those skilled in the art. Examples ofthese techniques are found in, for example, Berger et al., Guide toMolecular Cloning Techniques, Methods in Enzymology 152, Academic Press,Inc., San Diego, Calif. (Berger), which is incorporated herein byreference in its entirety.

Automated sequencing methods can be used to obtain or verify thenucleotide sequence of MMP. The MMP nucleotide sequences of the presentinvention are believed to be 100% accurate. However, as is known in theart, nucleotide sequence obtained by automated methods may contain someerrors. Nucleotide sequences determined by automation are typically atleast about 90%, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of a given nucleicacid molecule. The actual sequence may be more precisely determinedusing manual sequencing methods, which are well known in the art. Anerror in a sequence which results in an insertion or deletion of one ormore nucleotides may result in a frame shift in translation such thatthe predicted amino acid sequence will differ from that which would bepredicted from the actual nucleotide sequence of the nucleic acidmolecule, starting at the point of the mutation.

The nucleic acid molecules of the present invention, and fragmentsderived therefrom, are useful for screening for restriction fragmentlength polymorphism (RFLP) associated with certain disorders, as well asfor genetic mapping.

The polynucleotide sequence information provided by the invention makespossible large-scale expression of the encoded polypeptide by techniqueswell known and routinely practiced in the art.

Vectors

Another aspect of the present invention is directed to vectors, orrecombinant expression vectors, comprising any of the nucleic acidmolecules described above. Vectors are used herein either to amplify DNAor RNA encoding MMP and/or to express DNA which encodes MMP. Preferredvectors include, but are not limited to, plasmids, phages, cosmids,episomes, viral particles or viruses, and integratable DNA fragments(i.e., fragments integratable into the host genome by homologousrecombination). Preferred viral particles include, but are not limitedto, adenoviruses, baculoviruses, parvoviruses, herpesviruses,poxviruses, adeno-associated viruses, Semliki Forest viruses, vacciniaviruses, and retroviruses. Preferred expression vectors include, but arenot limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). Otherexpression vectors include, but are not limited to, pSPORT™ vectors,pGEM™ vectors (Promega), pPROEXvectors™ (LTI, Bethesda, Md.),Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™(Invitrogen), and pYES2™(Invitrogen).

Expression constructs preferably comprise MMP-encoding polynucleotidesoperatively linked to an endogenous or exogenous expression control DNAsequence and a transcription terminator. Expression control DNAsequences include promoters, enhancers, operators, and regulatoryelement binding sites generally, and are typically selected based on theexpression systems in which the expression construct is to be utilized.Preferred promoter and enhancer sequences are generally selected for theability to increase gene expression, while operator sequences aregenerally selected for the ability to regulate gene expression.Expression constructs of the invention may also include sequencesencoding one or more selectable markers that permit identification ofhost cells bearing the construct. Expression constructs may also includesequences that facilitate, and preferably promote, homologousrecombination in a host cell. Preferred constructs of the invention alsoinclude sequences necessary for replication in a host cell.

Expression constructs are preferably utilized for production of anencoded protein, but may also be utilized simply to amplify anMMP-encoding polynucleotide sequence. In preferred embodiments, thevector is an expression vector wherein the polynucleotide of theinvention is operatively linked to a polynucleotide comprising anexpression control sequence. Autonomously replicating recombinantexpression constructs such as plasmid and viral DNA vectorsincorporating polynucleotides of the invention are also provided.Preferred expression vectors are replicable DNA constructs in which aDNA sequence encoding MMP is operably linked or connected to suitablecontrol sequences capable of effecting the expression of the MMP in asuitable host. DNA regions are operably linked or connected when theyare functionally related to each other. For example, a promoter isoperably linked or connected to a coding sequence if it controls thetranscription of the sequence. Amplification vectors do not requireexpression control domains, but rather need only the ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants. The needfor control sequences in the expression vector will vary depending uponthe host selected and the transformation method chosen. Generally,control sequences include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding and sequences which control the termination oftranscription and translation.

Preferred vectors preferably contain a promoter that is recognized bythe host organism. The promoter sequences of the present invention maybe prokaryotic, eukaryotic or viral. Examples of suitable prokaryoticsequences include the P_(R) and P_(L) promoters of bacteriophage lambda(The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1973), which is incorporated herein byreference in its entirety; Lambda II, Hendrix, R. W., Ed., Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1980), which is incorporatedherein by reference in its entirety); the trp, recA, heat shock, andlacZ promoters of E. coli and the SV40 early promoter (Benoist et al.Nature, 1981, 290, 304-310, which is incorporated herein by reference inits entirety). Additional promoters include, but are not limited to,mouse mammary tumor virus, long terminal repeat of humanimmunodeficiency virus, maloney virus, cytomegalovirus immediate earlypromoter, Epstein Barr virus, Rous sarcoma virus, human actin, humanmyosin, human hemoglobin, human muscle creatine, and humanmetalothionein.

Additional regulatory sequences can also be included in preferredvectors. Preferred examples of suitable regulatory sequences arerepresented by the Shine-Dalgarno of the replicase gene of the phageMS-2 and of the gene cII of bacteriophage lambda. The Shine-Dalgarnosequence may be directly followed by DNA encoding MMP and result in theexpression of the mature MMP protein.

Moreover, suitable expression vectors can include an appropriate markerthat allows the screening of the transformed host cells. Thetransformation of the selected host is carried out using any one of thevarious techniques well known to the expert in the art and described inSambrook et al., supra.

An origin of replication can also be provided either by construction ofthe vector to include an exogenous origin or may be provided by the hostcell chromosomal replication mechanism. If the vector is integrated intothe host cell chromosome, the latter may be sufficient. Alternatively,rather than using vectors which contain viral origins of replication,one skilled in the art can transform mammalian cells by the method ofco-transformation with a selectable marker and MMP DNA. An example of asuitable marker is dihydrofolate reductase (DHFR) or thymidine kinase(see, U.S. Pat. No. 4,399,216).

Nucleotide sequences encoding MMP may be recombined with vector DNA inaccordance with conventional techniques, including blunt-ended orstaggered-ended termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesiderable joining, andligation with appropriate ligases. Techniques for such manipulation aredisclosed by Sambrook et al., supra and are well known in the art.Methods for construction of mammalian expression vectors are disclosedin, for example, Okayama et al., Mol. Cell. Biol., 1983, 3, 280, Cosmanet al., Mol. Immunol., 1986, 23, 935, Cosman et al., Nature, 1984, 312,768, EP-A-0367566, and WO 91/18982, each of which is incorporated hereinby reference in its entirety.

Host Cells

According to another aspect of the invention, host cells are provided,including prokaryotic and eukaryotic cells, comprising a polynucleotideof the invention (or vector of the invention) in a manner that permitsexpression of the encoded MMP polypeptide. Polynucleotides of theinvention may be introduced into the host cell as part of a circularplasmid, or as linear DNA comprising an isolated protein coding regionor a viral vector. Methods for introducing DNA into the host cell thatare well known and routinely practiced in the art includetransformation, transfection, electroporation, nuclear injection, orfusion with carriers such as liposomes, micelles, ghost cells, andprotoplasts. Expression systems of the invention include bacterial,yeast, fungal, plant, insect, invertebrate, vertebrate, and mammaliancells systems.

The invention provides host cells that are transformed or transfected(stably or transiently) with polynucleotides of the invention or vectorsof the invention. As stated above, such host cells are useful foramplifying the polynucleotides and also for expressing the MMPpolypeptide or fragment thereof encoded by the polynucleotide.

In still another related embodiment, the invention provides a method forproducing a MMP polypeptide (or fragment thereof) comprising the stepsof growing a host cell of the invention in a nutrient medium andisolating the polypeptide or variant thereof from the cell or themedium.

According to some aspects of the present invention, transformed hostcells having an expression vector comprising any of the nucleic acidmolecules described above are provided. Expression of the nucleotidesequence occurs when the expression vector is introduced into anappropriate host cell. Suitable host cells for expression of thepolypeptides of the invention include, but are not limited to,prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vectoris employed, then the appropriate host cell would be any prokaryoticcell capable of expressing the cloned sequences. Suitable prokaryoticcells include, but are not limited to, bacteria of the generaEscherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, andStaphylococcus.

If an eukaryotic expression vector is employed, then the appropriatehost cell would be any eukaryotic cell capable of expressing the clonedsequence. Preferably, eukaryotic cells are cells of higher eukaryotes.Suitable eukaryotic cells include, but are not limited to, non-humanmammalian tissue culture cells and human tissue culture cells. Preferredhost cells include, but are not limited to, insect cells, HeLa cells,Chinese hamster ovary cells (CHO cells), African green monkey kidneycells (COS cells), human HEK-293 cells, and murine 3T3 fibroblasts.Propagation of such cells in cell culture has become a routine procedure(see, Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973),which is incorporated herein by reference in its entirety).

In addition, a yeast host may be employed as a host cell. Preferredyeast cells include, but are not limited to, the genera Saccharomyces,Pichia, and Kluveromyces. Preferred yeast hosts are S. cerevisiae and P.pastoris. Preferred yeast vectors can contain an origin of replicationsequence from a 2T yeast plasmid, an autonomously replication sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Shuttle vectorsfor replication in both yeast and E. coli are also included herein.

Alternatively, insect cells may be used as host cells. In a preferredembodiment, the polypeptides of the invention are expressed using abaculovirus expression system (see, Luckow et al., Bio/Technology, 1988,6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O'Rielly etal. (Eds.), W. H. Freeman and Company, New York, 1992, and U.S. Pat. No.4,879,236, each of which is incorporated herein by reference in itsentirety). In addition, the MAXBAC™ complete baculovirus expressionsystem (Invitrogen) can, for example, be used for production in insectcells.

Host cells of the invention are a valuable source of immunogen fordevelopment of antibodies specifically immunoreactive with MMP. Hostcells of the invention are also useful in methods for the large-scaleproduction of MMP polypeptides wherein the cells are grown in a suitableculture medium and the desired polypeptide products are isolated fromthe cells, or from the medium in which the cells are grown, bypurification methods known in the art, e.g., conventionalchromatographic methods including immunoaffinity chromatography,affinity chromatography, hydrophobic interaction chromatography, lectinaffinity chromatography, size exclusion filtration, cation or anionexchange chromatography, high pressure liquid chromatography (HPLC),reverse phase HPLC, and the like. Still other methods of purificationinclude those methods wherein the desired protein is expressed andpurified as a fusion protein having a specific tag, label, or chelatingmoiety that is recognized by a specific binding partner or agent. Thepurified protein can be cleaved to yield the desired protein, or can beleft as an intact fusion protein. Cleavage of the fusion component mayproduce a form of the desired protein having additional amino acidresidues as a result of the cleavage process.

Knowledge of MMP DNA sequences allows for modification of cells topermit, or increase, expression of endogenous MMP. Cells can be modified(e.g., by homologous recombination) to provide increased expression byreplacing, in whole or in part, the naturally occurring MMP promoterwith all or part of a heterologous promoter so that the cells expressMMP at higher levels. The heterologous promoter is inserted in such amanner that it is operatively linked to endogenous MMP encodingsequences. (See, for example, PCT International Publication No. WO94/12650, PCT International Publication No. WO 92/20808, and PCTInternational Publication No. WO 91/09955.) It is also contemplatedthat, in addition to heterologous promoter DNA, amplifiable marker DNA(e.g., ada, dhfr, and the multifunctional CAD gene which encodescarbamoyl phosphate synthase, aspartate transcarbamylase, anddihydroorotase) and/or intron DNA may be inserted along with theheterologous promoter DNA. If linked to the MMP coding sequence,amplification of the marker DNA by standard selection methods results inco-amplification of the MMP coding sequences in the cells.

Knock-outs

The DNA sequence information provided by the present invention alsomakes possible the development (e.g., by homologous recombination or“knock-out” strategies; see Capecchi, Science 244:1288-1292 (1989),which is incorporated herein by reference) of animals that fail toexpress functional MMP or that express a variant of MMP. Such animals(especially small laboratory animals such as rats, rabbits, and mice)are useful as models for studying the in vivo activities of MMP andmodulators of MMP.

Antisense

Also made available by the invention are anti-sense polynucleotides thatrecognize and hybridize to polynucleotides encoding NMP. Full-length andfragment anti-sense polynucleotides are provided. Fragment antisensemolecules of the invention include (i) those that specifically recognizeand hybridize to MMP RNA (as determined by sequence comparison of DNAencoding MMP to DNA encoding other known molecules). Identification ofsequences unique to MMP encoding polynucleotides can be deduced throughuse of any publicly available sequence database, and/or through use ofcommercially available sequence comparison programs. Afteridentification of the desired sequences, isolation through restrictiondigestion or amplification using any of the various polymerase chainreaction techniques well known in the art can be performed. Anti-sensepolynucleotides are particularly relevant to regulating expression ofMMP by those cells expressing MMP mRNA.

Antisense nucleic acids (preferably 10 to 30 base-pair oligonucleotides)capable of specifically binding to MMP expression control sequences orMMP RNA are introduced into cells (e.g., by a viral vector or colloidaldispersion system such as a liposome). The antisense nucleic acid bindsto the MMP target nucleotide sequence in the cell and preventstranscription and/or translation of the target sequence.Phosphorothioate and methylphosphonate antisense oligonucleotides arespecifically contemplated for therapeutic use by the invention. Theantisense oligonucleotides may be further modified by addingpoly-L-lysine, transferrin polylysine, or cholesterol moieties at their5′ end. Suppression of MMP expression at either the transcriptional ortranslational level is useful to generate cellular or animal models fordiseases/conditions characterized by aberrant MMP expression.

Antisense oligonucleotides, or fragments of sequences selected from thegroup consisting of SEQ ID NO:1 to SEQ ID NO:3, or sequencescomplementary or homologous thereto, derived from the nucleotidesequences of the present invention encoding MMP are useful as diagnostictools for probing gene expression in various tissues. For example,tissue can be probed in situ with oligonucleotide probes carryingdetectable groups by conventional autoradiography techniques toinvestigate native expression of this enzyme or pathological conditionsrelating thereto. Antisense oligonucleotides are preferably directed toregulatory regions of sequences selected from the group consisting ofSEQ ID NO:1 to SEQ ID NO:3, or mRNA corresponding thereto, including,but not limited to, the initiation codon, TATA box, enhancer sequences,and the like.

Transcription Factors

The MMP sequences taught in the present invention facilitate the designof novel transcription factors for modulating MMP expression in nativecells and animals, and cells transformed or transfected with MMPpolynucleotides. For example, the Cys₂-His₂ zinc finger proteins, whichbind DNA via their zinc finger domains, have been shown to be amenableto structural changes that lead to the recognition of different targetsequences. These artificial zinc finger proteins recognize specifictarget sites with high affinity and low dissociation constants, and areable to act as gene switches to modulate gene expression. Knowledge ofthe particular MMP target sequence of the present invention facilitatesthe engineering of zinc finger proteins specific for the target sequenceusing known methods such as a combination of structure-based modelingand screening of phage display libraries (Segal et al., Proc. Natl.Acad. Sci. (USA) 96:2758-2763 (1999); Liu et al., Proc. Natl. Acad. Sci.(USA) 94:5525-5530 (1997); Greisman et al., Science 275:657-661 (1997);Choo et al., J. Mol. Biol. 273:525-532 (1997)). Each zinc finger domainusually recognizes three or more base pairs. Since a recognitionsequence of 18 base pairs is generally sufficient in length to render itunique in any known genome, a zinc finger protein consisting of 6 tandemrepeats of zinc fingers would be expected to ensure specificity for aparticular sequence (Segal et al.) The artificial zinc finger repeats,designed based on MMP sequences, are fused to activation or repressiondomains to promote or suppress MMP expression (Liu et al.)Alternatively, the zinc finger domains can be fused to the TATAbox-binding factor (TBP) with varying lengths of linker region betweenthe zinc finger peptide and the TBP to create either transcriptionalactivators or repressors (Kim et al., Proc. Natl. Acad. Sci. (USA)94:3616-3620 (1997). Such proteins and polynucleotides that encode them,have utility for modulating MMP expression in vivo in both native cells,animals and humans; and/or cells transfected with MMP-encodingsequences. The novel transcription factor can be delivered to the targetcells by transfecting constructs that express the transcription factor(gene therapy), or by introducing the protein. Engineered zinc fingerproteins can also be designed to bind RNA sequences for use intherapeutics as alternatives to antisense or catalytic RNA methods(McColl et al., Proc. Natl. Acad. Sci. (USA) 96:9521-9526 (1997); Wu etal., Proc. Natl. Acad. Sci. (USA) 92:344-348 (1995)). The presentinvention contemplates methods of designing such transcription factorsbased on the gene sequence of the invention, as well as customized zincfinger proteins, that are useful to modulate MMP expression in cells(native or transformed) whose genetic complement includes thesesequences.

Polypeptides

The invention also provides purified and isolated mammalian MMPpolypeptides encoded by a polynucleotide of the invention. Presentlypreferred is a human MMP polypeptide comprising the amino acid sequenceset out in sequences selected from the group consisting of SEQ ID NO:4to SEQ ID NO:6, or fragments thereof comprising an epitope specific tothe polypeptide. By “epitope specific to” is meant a portion of the MMPthat is recognizable by an antibody that is specific for the MMP, asdefined in detail below.

Although the sequences provided are particular human sequences, theinvention is intended to include within its scope other human allelicvariants; non-human mammalian forms of MMP, and other vertebrate formsof MMP.

The invention also embraces polypeptides that have at least 99%, atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55% or at least 50%identity and/or homology to the preferred polypeptide of the invention.Percent amino acid sequence “identity” with respect to the preferredpolypeptide of the invention is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical withthe residues in the MMP sequence after aligning both sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Percent sequence “homology” with respect to thepreferred polypeptide of the invention is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the residues in the MMP sequence after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and also considering any conservativesubstitutions as part of the sequence identity.

In one aspect, percent homology is calculated as the percentage of aminoacid residues in the smaller of two sequences which align with identicalamino acid residue in the sequence being compared, when four gaps in alength of 100 amino acids may be introduced to maximize alignment(Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124,National Biochemical Research Foundation, Washington, D.C. (1972),incorporated herein by reference).

Polypeptides of the invention may be isolated from natural cell sourcesor may be chemically synthesized, but are preferably produced byrecombinant procedures involving host cells of the invention. Use ofmammalian host cells is expected to provide for such post-translationalmodifications (e.g., glycosylation, truncation, lipidation, andphosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention. Glycosylated andnon-glycosylated forms of MMP polypeptides are embraced by theinvention.

The invention also embraces variant (or analog) MMP polypeptides. In oneexample, insertion variants are provided wherein one or more amino acidresidues supplement a MMP amino acid sequence. Insertions may be locatedat either or both termini of the protein, or may be positioned withininternal regions of the MMP amino acid sequence. Insertional variantswith additional residues at either or both termini can include, forexample, fusion proteins and proteins including amino acid tags orlabels.

Insertion variants include MMP polypeptides wherein one or more aminoacid residues are added to a MMP acid sequence or to a biologicallyactive fragment thereof.

Variant products of the invention also include mature MMP products,i.e., MMP products wherein leader or signal sequences are removed, withadditional amino terminal residues. The additional amino terminalresidues may be derived from another protein, or may include one or moreresidues that are not identifiable as being derived from specificproteins. MMP products with an additional methionine residue at position−1 (Met⁻¹-MMP) are contemplated, as are variants with additionalmethionine and lysine residues at positions −2 and −1 (Met⁻²-Lys⁻¹-MMP).Variants of MMP with additional Met, Met-Lys, Lys residues (or one ormore basic residues in general) are particularly useful for enhancedrecombinant protein production in bacterial host cells.

The invention also embraces MMP variants having additional amino acidresidues that result from use of specific expression systems. Forexample, use of commercially available vectors that express a desiredpolypeptide as part of a glutathione-S-transferase (GST) fusion productprovides the desired polypeptide having an additional glycine residue atposition −1 after cleavage of the GST component from the desiredpolypeptide. Variants that result from expression in other vectorsystems are also contemplated.

Insertional variants also include fusion proteins wherein the aminoterminus and/or the carboxy terminus of MMP is/are fused to anotherpolypeptide.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a MMP polypeptide are removed. Deletionscan be effected at one or both termini of the MMP polypeptide, or withremoval of one or more non-terminal amino acid residues of MMP. Deletionvariants, therefore, include all fragments of a MMP polypeptide.

The invention also embraces polypeptide fragments of sequences selectedfrom the group consisting of SEQ ID NO:4 to SEQ ID NO:6, wherein thefragments maintain biological (e.g., proteinase or collagenase activity)and immunological properties of a MMP polypeptide.

In one preferred embodiment of the invention, an isolated nucleic acidmolecule comprises a nucleotide sequence that encodes a polypeptidecomprising an amino acid sequence homologous to sequences selected fromthe group consisting of SEQ ID NO:4 to SEQ ID NO:6, and fragmentsthereof, wherein the nucleic acid molecule encoding at least a portionof MMP. In a more preferred embodiment, the isolated nucleic acidmolecule comprises a sequence that encodes a polypeptide comprisingsequences selected from the group consisting of SEQ ID NO:4 to SEQ EDNO:6, and fragments thereof.

One preferred embodiment of the present invention provides an isolatednucleic acid molecule comprising a sequence homologous sequencesselected from the group consisting of SEQ ID NO:1 to SEQ ID NO:3, andfragments thereof. Another preferred embodiment provides an isolatednucleic acid molecule comprising a sequence selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:3, and fragments thereof.

In a preferred embodiment, the isolated nucleic acid molecule comprisesa nucleotide sequence which encodes a polypeptide comprising a sequenceof SEQ ID NO:5, or a fragment thereof. In a more preferred embodiment,the polypeptide encoded by the nucleotide sequence does not have thesequence of SEQ ID NO:8.

As used in the present invention, polypeptide fragments comprise atleast 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids ofsequences selected from the group consisting of SEQ ID NO:4 to SEQ IDNO:6. Preferred polypeptide fragments display antigenic propertiesunique to, or specific for, human MMP and its allelic and specieshomologs. Fragments of the invention having the desired biological andimmunological properties can be prepared by any of the methods wellknown and routinely practiced in the art.

In still another aspect, the invention provides substitution variants ofMMP polypeptides. Substitution variants include those polypeptideswherein one or more amino acid residues of a MMP polypeptide are removedand replaced with alternative residues. In one aspect, the substitutionsare conservative in nature; however, the invention embracessubstitutions that are also non-conservative. Conservative substitutionsfor this purpose may be defined as set out in Tables 2, 3, or 4 below.

Variant polypeptides include those wherein conservative substitutionshave been introduced by modification of polynucleotides encodingpolypeptides of the invention. Amino acids can be classified accordingto physical properties and contribution to secondary and tertiaryprotein structure. A conservative substitution is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions are set out inTable 2 (from WO 97/09433, page 10, published Mar. 13, 1997(PCT/GB96/02197, filed Sep. 6, 1996), immediately below.

TABLE 2 Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINOACID Aliphatic Non-polar G A P I L V Polar - uncharged C S T M N QPolar - charged D E K R Aromatic H F W Y Other N Q D E

Alternatively, conservative amino acids can be grouped as described inLehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY,N.Y. (1975), pp.71-77] as set out in Table 3, below.

TABLE 3 Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D E

As still another alternative, exemplary conservative substitutions areset out in Table 4, below.

TABLE 4 Conservative Substitutions III Original Residue ExemplarySubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

It should be understood that the definition of polypeptides of theinvention is intended to include polypeptides bearing modificationsother than insertion, deletion, or substitution of amino acid residues.By way of example, the modifications may be covalent in nature, andinclude for example, chemical bonding with polymers, lipids, otherorganic, and inorganic moieties. Such derivatives may be prepared toincrease circulating half-life of a polypeptide, or may be designed toimprove the targeting capacity of the polypeptide for desired cells,tissues, or organs. Similarly, the invention further embraces MMPpolypeptides that have been covalently modified to include one or morewater-soluble polymer attachments such as polyethylene glycol,polyoxyethylene glycol, or polypropylene glycol. Variants that displayligand binding properties of native MMP and are expressed at higherlevels are particularly useful in assays of the invention; the variantsare also useful in providing cellular, tissue and animal models ofdiseases/conditions characterized by aberrant MMP activity.

In a related embodiment, the present invention provides compositionscomprising purified polypeptides of the invention. Preferredcompositions comprise, in addition to the polypeptide of the invention,a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid,semisolid, or solid diluent that serves as a pharmaceutical vehicle,excipient, or medium. Any diluent known in the art may be used.Exemplary diluents include, but are not limited to, water, salinesolutions, polyoxyethylene sorbitan monolaurate, magnesium stearate,methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose,sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate,mineral oil, and cocoa butter.

Antibodies

Also comprehended by the present invention are antibodies (e.g.,monoclonal and polyclonal antibodies, single chain antibodies, chimericantibodies, bifunctional/bispecific antibodies, humanized antibodies,human antibodies, and complementary determining region (CDR)-graftedantibodies, including compounds which include CDR sequences whichspecifically recognize a polypeptide of the invention) specific for MMPor fragments thereof. Preferred antibodies of the invention are humanantibodies that are produced and identified according to methodsdescribed in WO93/11236, published Jun. 20, 1993, which is incorporatedherein by reference in its entirety. Antibody fragments, including Fab,Fab′, F(ab′)₂, and F_(v), are also provided by the invention. The term“specific for,” when used to describe antibodies of the invention,indicates that the variable regions of the antibodies of the inventionrecognize and bind MMP polypeptides exclusively (i.e., are able todistinguish MMP polypeptides from other known MMP polypeptides by virtueof measurable differences in binding affinity, despite the possibleexistence of localized sequence identity, homology, or similaritybetween MMP and such polypeptides). It will be understood that specificantibodies may also interact with other proteins (for example, S. aureusprotein A or other antibodies in ELISA techniques) through interactionswith sequences outside the variable region of the antibodies, and, inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of an antibody of the invention are wellknown and routinely practiced in the art. For a comprehensive discussionof such assays, see Harlow et al. (Eds.), Antibodies A LaboratoryManual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988),Chapter 6. Antibodies that recognize and bind fragments of the MMPpolypeptides of the invention are also contemplated, provided that theantibodies are specific for MMP polypeptides. Antibodies of theinvention can be produced using any method well known and routinelypracticed in the art.

The invention provides an antibody that is specific for the MMP of theinvention. Antibody specificity is described in greater detail below.However, it should be emphasized that antibodies that can be generatedfrom polypeptides that have previously been described in the literatureand that are capable of fortuitously cross-reacting with MMP (e.g., dueto the fortuitous existence of a similar epitope in both polypeptides)are considered “cross-reactive” antibodies. Such cross-reactiveantibodies are not antibodies that are “specific” for MMP. Thedetermination of whether an antibody is specific for MMP or iscross-reactive with another known MMP is made using any of severalassays, such as Western blotting assays, that are well known in the art.

In one preferred variation, the invention provides monoclonalantibodies. Hybridomas that produce such antibodies also are intended asaspects of the invention. In yet another variation, the inventionprovides a humanized antibody. Humanized antibodies are useful for invivo therapeutic indications.

In another variation, the invention provides a cell-free compositioncomprising polyclonal antibodies, wherein at least one of the antibodiesis an antibody of the invention specific for MMP. Antisera isolated froman animal is an exemplary composition, as is a composition comprising anantibody fraction of an antisera that has been resuspended in water orin another diluent, excipient, or carrier.

In still another related embodiment, the invention provides ananti-idiotypic antibody specific for an antibody that is specific forMMP.

It is well known that antibodies contain relatively small antigenbinding domains that can be isolated chemically or by recombinanttechniques. Such domains are useful MMP binding molecules themselves,and also may be reintroduced into human antibodies, or fused to toxinsor other polypeptides. Thus, in still another embodiment, the inventionprovides a polypeptide comprising a fragment of an MMP-specificantibody, wherein the fragment and the polypeptide bind to the MMP. Byway of non-limiting example, the invention provides polypeptides thatare single chain antibodies and CDR-grafted antibodies.

Non-human antibodies may be humanized by any of the methods known in theart. In one method, the non-human CDRs are inserted into a humanantibody or consensus antibody framework sequence. Further changes canthen be introduced into the antibody framework to modulate affinity orimmunogenicity.

Antibodies of the invention are useful for, e.g., therapeutic purposes(by modulating activity of MMP), diagnostic purposes to detect orquantitate MMP, and purification of MMP. Kits comprising an antibody ofthe invention for any of the purposes described herein are alsocomprehended. In general, a kit of the invention also includes a controlantigen for which the antibody is immunospecific.

Compositions

Mutations in the MMP gene that result in loss of normal function of theMMP gene product underlie MMP-related human disease states. Theinvention comprehends gene therapy to restore MMP activity to treatthose disease states. Delivery of a functional MMP gene to appropriatecells is effected ex vivo, in situ, or in vivo by use of vectors, andmore particularly viral vectors (e.g., adenovirus, adeno-associatedvirus, or a retrovirus), or ex vivo by use of physical DNA transfermethods (e.g., liposomes or chemical treatments). See, for example,Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). Foradditional reviews of gene therapy technology see Friedmann, Science,244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); andMiller, Nature, 357: 455-460 (1992). Alternatively, it is contemplatedthat in other human disease states, preventing the expression of, orinhibiting the activity of, MMP will be useful in treating diseasestates. It is contemplated that antisense therapy or gene therapy couldbe applied to negatively regulate the expression of MMP.

Another aspect of the present invention is directed to compositions,including pharmaceutical compositions, comprising any of the nucleicacid molecules or recombinant expression vectors described above and anacceptable carrier or diluent. Preferably, the carrier or diluent ispharmaceutically acceptable. Suitable carriers are described in the mostrecent edition of Remington's Pharnaceutical Sciences, A. Osol, astandard reference text in this field, which is incorporated herein byreference in its entirety. Preferred examples of such carriers ordiluents include, but are not limited to, water, saline, Ringer'ssolution, dextrose solution, and 5% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils may also be used. Theformulations are sterilized by commonly used techniques.

Also within the scope of the invention are compositions comprisingpolypeptides, polynucleotides, or antibodies of the invention that havebeen formulated with, e.g., a pharmaceutically acceptable carrier.

The invention also provides methods of using antibodies of theinvention. For example, the invention provides a method for modulatingligand binding of a MMP comprising the step of contacting the MMP withan antibody specific for the MMP, under conditions wherein the antibodybinds the MMP.

As discussed above, it is well known that MMPs are expressed in manydifferent tissues and regions, including in the brain. MMPs that may beexpressed in the brain provide an indication that aberrant MMP activitymay correlate with one or more neurological or psychological disorders.The invention also provides a method for treating a neurological orpsychiatric disorder comprising the step of administering to a mammal inneed of such treatment an amount of an antibody-like polypeptide of theinvention that is sufficient to modulate ligand binding to a MMP inneurons of the mammal. MMP may also be expressed in other tissues,including but not limited to, including but not limited to pancreas (andparticularly pancreatic islet tissue), pituitary, skeletal muscle,adipose tissue, liver, and thyroid, and may be found in many othertissues.

Kits

The present invention is also directed to kits, including pharmaceuticalkits. The kits can comprise any of the nucleic acid molecules describedabove, any of the polypeptides described above, or any antibody whichbinds to a polypeptide of the invention as described above, as well as anegative control. The kit preferably comprises additional components,such as, for example, instructions, solid support, reagents helpful forquantification, and the like.

In another aspect, the invention features methods for detection of apolypeptide in a sample as a diagnostic tool for diseases or disorders,wherein the method comprises the steps of: (a) contacting the samplewith a nucleic acid probe which hybridizes under hybridization assayconditions to a nucleic acid target region of a polypeptide havingsequences selected from the group consisting of SEQ ID NO:4 to SEQ IDNO:6, said probe comprising the nucleic acid sequence encoding thepolypeptide, fragments thereof, and the complements of the sequences andfragments; and (b) detecting the presence or amount of the probe:targetregion hybrid as an indication of the disease.

In preferred embodiments of the invention, the disease is selected fromthe group consisting of metabolic diseases and disorders (e.g., type 2diabetes, obesity, cardiovascular, dyslipidemias, adipogenesis,retinopathies, neuropathies, nephropathies etc.), proliferative diseasesand cancers (e.g., different cancers such as breast, colon, lung, etc.,tumor growth, tumor invasion, and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc.), hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.), CNS disorders (e.g., degenerative disorders such as Parkinson's,Alzheimer's, etc.), inflammatory conditions (e.g., Chron's disease,arthritis), diseases related to cell differentiation and homeostasis,cardiomyopathy, atherosclerosis, thromboembolic diseases, Sjögren'ssyndrome, renal failure, periodontal diseases, retinalneovascularization, wound healing, and neurodegenerative diseasesincluding, for example, Alzheimer's disease, multiple sclerosis,Parkinson's disease, and motoneuron disease, among others.

Kits may be designed to detect either expression of polynucleotidesencoding MMP expressed in the brain or the MMP proteins themselves inorder to identify tissue as being neurological. For example,oligonucleotide hybridization kits can be provided which include acontainer having an oligonucleotide probe specific for the MMP-specificDNA and optionally, containers with positive and negative controlsand/or instructions. Similarly, PCR kits can be provided which include acontainer having primers specific for the NMP-specific sequences, DNAand optionally, containers with size markers, positive and negativecontrols and/or instructions.

Hybridization conditions should be such that hybridization occurs onlywith the genes in the presence of other nucleic acid molecules. Understringent hybridization conditions only highly complementary nucleicacid sequences hybridize. Preferably, such conditions preventhybridization of nucleic acids having 1 or 2 mismatches out of 20contiguous nucleotides. Such conditions are defined supra.

The diseases for which detection of genes in a sample could bediagnostic include diseases in which nucleic acid (DNA and/or RNA) isamplified in comparison to normal cells. By “amplification” is meantincreased numbers of DNA or RNA in a cell compared with normal cells.

The diseases that could be diagnosed by detection of nucleic acid in asample preferably include central nervous system and metabolic diseases.The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The samples used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample that is compatiblewith the method utilized.

Alternatively, immunoassay kits can be provided which have containerscontainer having antibodies specific for the MMP-protein and optionally,containers with positive and negative controls and/or instructions.

Kits may also be provided useful in the identification of MMP bindingpartners such as natural ligands or modulators (agonists orantagonists). Substances useful for treatment of disorders or diseasespreferably show positive results in one or more in vitro assays for anactivity corresponding to treatment of the disease or disorder inquestion. Substances that modulate the activity of the polypeptidespreferably include, but are not limited to, antisense oligonucleotides,agonists and antagonists, and inhibitors of protein kinases.

Methods of Inducing Immune Response

Another aspect of the present invention is directed to methods ofinducing an immune response in a mammal against a polypeptide of theinvention by administering to the mammal an amount of the polypeptidesufficient to induce an immune response. The amount will be dependent onthe animal species, size of the animal, and the like but can bedetermined by those skilled in the art.

Methods of Identifying Ligands

The invention also provides assays to identify compounds that bind MMP.One such assay comprises the steps of: (a) contacting a compositioncomprising a MMP with a compound suspected of binding MMP; and (b)measuring binding between the compound and MMP. In one variation, thecomposition comprises a cell expressing MMP on its surface. In anothervariation, isolated MMP or cell membranes comprising MMP are employed.The binding may be measured directly, e.g., by using a labeled compound,or may be measured indirectly. Following steps (a) and (b), compoundsidentified as binding MMP may be tested in other assays including, butnot limited to, in vivo models, to confirm or quantitate binding to MMP.

Specific binding molecules, including natural ligands and syntheticcompounds, can be identified or developed using isolated or recombinantMMP products, MMP variants, or preferably, cells expressing suchproducts. Binding partners are useful for purifying MMP products anddetection or quantification of MMP products in fluid and tissue samplesusing known immunological procedures. Binding molecules are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) biological activities of MMP, especially those activitiesinvolved in collagenase or proteinase activity.

The DNA and amino acid sequence information provided by the presentinvention also makes possible identification of binding partnercompounds with which an MMP polypeptide or polynucleotide will interact.Methods to identify binding partner compounds include solution assays,in vitro assays wherein MMP polypeptides are immobilized, and cell-basedassays. Identification of binding partner compounds of MMP polypeptidesprovides candidates for therapeutic or prophylactic intervention inpathologies associated with MMP normal and aberrant biological activity.

The invention includes several assay systems for identifying MMP bindingpartners. In solution assays, methods of the invention comprise thesteps of (a) contacting a MMP polypeptide with one or more candidatebinding partner compounds and (b) identifying the compounds that bind tothe MMP polypeptide. Identification of the compounds that bind the MMPpolypeptide can be achieved by isolating the MMP polypeptide/bindingpartner complex, and separating the binding partner compound from theMMP polypeptide. An additional step of characterizing the physical,biological, and/or biochemical properties of the binding partnercompound is also comprehended in another embodiment of the invention,wherein compounds identified as binding MMP may be tested in otherassays including, but not limited to, in vivo models, to confirm orquantitate binding to MMP. In one aspect, the MMP polypeptide/bindingpartner complex is isolated using an antibody immunospecific for eitherthe MMP polypeptide or the candidate binding partner compound.

In still other embodiments, either the MMP polypeptide or the candidatebinding partner compound comprises a label or tag that facilitates itsisolation, and methods of the invention to identify binding partnercompounds include a step of isolating the MMP polypeptide/bindingpartner complex through interaction with the label or tag. An exemplarytag of this type is a poly-histidine sequence, generally around sixhistidine residues, that permits isolation of a compound so labeledusing nickel chelation. Other labels and tags, such as the FLAG® tag(Eastman Kodak, Rochester, N.Y.), well known and routinely used in theart, are embraced by the invention.

In one variation of an in vitro assay, the invention provides a methodcomprising the steps of (a) contacting an immobilized MMP polypeptidewith a candidate binding partner compound and (b) detecting binding ofthe candidate compound to the MMP polypeptide. In an alternativeembodiment, the candidate binding partner compound is immobilized andbinding of MMP is detected. Immobilization is accomplished using any ofthe methods well known in the art, including covalent bonding to asupport, a bead, or a chromatographic resin, as well as non-covalent,high affinity interactions such as antibody binding, or use ofstreptavidin/biotin binding wherein the immobilized compound includes abiotin moiety. Detection of binding can be accomplished (i) using aradioactive label on the compound that is not immobilized, (ii) using ofa fluorescent label on the non-immobilized compound, (iii) using anantibody immunospecific for the non-immobilized compound, (iv) using alabel on the non-immobilized compound that excites a fluorescent supportto which the immobilized compound is attached, as well as othertechniques well known and routinely practiced in the art.

Another aspect of the present invention is directed to methods ofidentifying compounds that bind to either MMP or nucleic acid moleculesencoding MMP, comprising contacting MMP, or a nucleic acid moleculeencoding the same, with a compound, and determining whether the compoundbinds MMP or a nucleic acid molecule encoding the same. Binding can bedetermined by binding assays which are well known to the skilledartisan, including, but not limited to, gel-shift assays, Western blots,radiolabeled competition assay, phage-based expression cloning,co-fractionation by chromatography, co-precipitation, cross linking,interaction trap/two-hybrid analysis, southwestern analysis, ELISA, andthe like, which are described in, for example, Current Protocols inMolecular Biology, 1999, John Wiley & Sons, NY, which is incorporatedherein by reference in its entirety. The compounds to be screenedinclude (which may include compounds which are suspected to bind MMP, ora nucleic acid molecule encoding the same), but are not limited to,extracellular, intracellular, biologic or chemical origin. The methodsof the invention also embrace ligands, especially neuropeptides, thatare attached to a label, such as a radiolabel (e.g., ¹²⁵I, ³⁵S, ³²P,³³P, ³H), a fluorescence label, a chemiluminescent label, an enzymiclabel and an immunogenic label. Modulators falling within the scope ofthe invention include, but are not limited to, non-peptide moleculessuch as non-peptide mimetics, non-peptide allosteric effectors, andpeptides. The MMP polypeptide or polynucleotide employed in such a testmay either be free in solution, attached to a solid support, borne on acell surface or located intracellularly or associated with a portion ofa cell. One skilled in the art can, for example, measure the formationof complexes between MMP and the compound being tested. Alternatively,one skilled in the art can examine the diminution in complex formationbetween MMP and its substrate caused by the compound being tested.

In another embodiment of the invention, high throughput screening forcompounds having suitable binding affinity to MMP is employed. Briefly,large numbers of different test compounds are synthesized on a solidsubstrate. The peptide test compounds are contacted with MMP and washed.Bound MMP is then detected by methods well known in the art. Purifiedpolypeptides of the invention can also be coated directly onto platesfor use in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the protein andimmobilize it on the solid support.

Generally, an expressed MMP can be used for HTS binding assays inconjunction with its defined ligand, in this case the correspondingneuropeptide that activates it. The identified peptide is labeled with asuitable radioisotope, including, but not limited to, ¹²⁵I, ³H, ³⁵S or³²P, by methods that are well known to those skilled in the art.Alternatively, the peptides may be labeled by well-known methods with asuitable fluorescent derivative (Baindur et al., Drug Dev. Res., 1994,33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160).Alternative methods include a scintillation proximity assay (SPA) or aFlashPlate format in which such separation is unnecessary (Nakayama,Cur. Opinion Drug Disc. Dev., 1998, 1, 85-91; Bosse et al., J.Biomolecular Screening, 1998, 3, 285-292.). Binding of fluorescentligands can be detected in various ways, including fluorescence energytransfer (FRET), direct spectrophotofluorometric analysis of boundligand, or fluorescence polarization (Rogers, Drug Discovery Today,1997, 2, 156-160; Hill, Cur. Opinion Drug Disc. Dev., 1998, 1, 92-97).

Other assays may be used to identify specific ligands of an MMP,including assays that identify ligands of the target protein throughmeasuring direct binding of test ligands to the target protein, as wellas assays that identify ligands of target proteins through affinityultrafiltration with ion spray mass spectroscopy/HPLC methods or otherphysical and analytical methods. Alternatively, such bindinginteractions are evaluated indirectly using the yeast two-hybrid systemdescribed in Fields et al., Nature, 340:245-246 (1989), and Fields etal., Trends in Genetics, 10:286-292 (1994), both of which areincorporated herein by reference. The two-hybrid system is a geneticassay for detecting interactions between two proteins or polypeptides.It can be used to identify proteins that bind to a known protein ofinterest, or to delineate domains or residues critical for aninteraction. Variations on this methodology have been developed to clonegenes that encode DNA binding proteins, to identify peptides that bindto a protein, and to screen for drugs. The two-hybrid system exploitsthe ability of a pair of interacting proteins to bring a transcriptionactivation domain into close proximity with a DNA binding domain thatbinds to an upstream activation sequence (UAS) of a reporter gene, andis generally performed in yeast. The assay requires the construction oftwo hybrid genes encoding (1) a DNA-binding domain that is fused to afirst protein and (2) an activation domain fused to a second protein.The DNA-binding domain targets the first hybrid protein to the UAS ofthe reporter gene; however, because most proteins lack an activationdomain, this DNA-binding hybrid protein does not activate transcriptionof the reporter gene. The second hybrid protein, which contains theactivation domain, cannot by itself activate expression of the reportergene because it does not bind the UAS. However, when both hybridproteins are present, the noncovalent interaction of the first andsecond proteins tethers the activation domain to the UAS, activatingtranscription of the reporter gene. For example, when the first proteinis a MMP gene product, or fragment thereof, that is known to interactwith another protein or nucleic acid, this assay can be used to detectagents that interfere with the binding interaction. Expression of thereporter gene is monitored as different test agents are added to thesystem. The presence of an inhibitory agent results in lack of areporter signal.

The yeast two-hybrid assay can also be used to identify proteins thatbind to the gene product. In an assay to identify proteins that bind toa MMP, or fragment thereof, a fusion polynucleotide encoding both a MMP(or fragment) and a UAS binding domain (i.e., a first protein) may beused. In addition, a large number of hybrid genes each encoding adifferent second protein fused to an activation domain are produced andscreened in the assay. Typically, the second protein is encoded by oneor more members of a total cDNA or genomic DNA fusion library, with eachsecond protein-coding region being fused to the activation domain. Thissystem is applicable to a wide variety of proteins, and it is not evennecessary to know the identity or function of the second bindingprotein. The system is highly sensitive and can detect interactions notrevealed by other methods; even transient interactions may triggertranscription to produce a stable mRNA that can be repeatedly translatedto yield the reporter protein.

Other assays may be used to search for agents that bind to the targetprotein. One such screening method to identify direct binding of testligands to a target protein is described in U.S. Pat. No. 5,585,277,incorporated herein by reference. This method relies on the principlethat proteins generally exist as a mixture of folded and unfoldedstates, and continually alternate between the two states. When a testligand binds to the folded form of a target protein (i.e., when the testligand is a ligand of the target protein), the target protein moleculebound by the ligand remains in its folded state. Thus, the folded targetprotein is present to a greater extent in the presence of a test ligandwhich binds the target protein, than in the absence of a ligand. Bindingof the ligand to the target protein can be determined by any method thatdistinguishes between the folded and unfolded states of the targetprotein. The function of the target protein need not be known in orderfor this assay to be performed. Virtually any agent can be assessed bythis method as a test ligand, including, but not limited to, metals,polypeptides, proteins, lipids, polysaccharides, polynucleotides andsmall organic molecules.

Another method for identifying ligands of a target protein is describedin Wieboldt et al., Anal. Chem., 69:1683-1691 (1997), incorporatedherein by reference. This technique screens combinatorial libraries of20-30 agents at a time in solution phase for binding to the targetprotein. Agents that bind to the target protein are separated from otherlibrary components by simple membrane washing. The specifically selectedmolecules that are retained on the filter are subsequently liberatedfrom the target protein and analyzed by HPLC and pneumatically assistedelectrospray (ion spray) ionization mass spectroscopy. This procedureselects library components with the greatest affinity for the targetprotein, and is particularly useful for small molecule libraries.

Other embodiments of the invention comprise using competitive screeningassays in which neutralizing antibodies capable of binding a polypeptideof the invention specifically compete with a test compound for bindingto the polypeptide. In this manner, the antibodies can be used to detectthe presence of any peptide that shares one or more antigenicdeterminants with MMP. Radiolabeled competitive binding studies aredescribed in A. H. Lin et al. Antimicrobial Agents and Chemotherapy,1997, vol. 41, no. 10. pp. 2127-2131, the disclosure of which isincorporated herein by reference in its entirety.

Identification of Modulating Agents

The invention also provides methods for identifying a modulator ofbinding between a MMP and a MMP binding partner, comprising the stepsof: (a) contacting a MMP binding partner and a composition comprising aMMP in the presence and in the absence of a putative modulator compound;(b) detecting binding between the binding partner and the a MMP; and (c)identifying a putative modulator compound or a modulator compound inview of decreased or increased binding between the binding partner andthe MMP in the presence of the putative modulator, as compared tobinding in the absence of the putative modulator. Following steps (a)and (b), compounds identified as modulating binding between MMP and anMMP binding partner may be tested in other assays including, but notlimited to, in vivo models, to confirm or quantitate modulation ofbinding to MMP.

MMP binding partners that stimulate MMP activity are useful as agonistsin disease states or conditions characterized by insufficient MMPactivity. e.g., as a result of insufficient activity of a MMP ligand).MMP binding partners that block ligand-mediated MMP signaling are usefulas MMP antagonists to treat disease states or conditions characterizedby excessive MMP signaling. In addition MMP modulators in general, aswell as MMP polynucleotides and polypeptides, are useful in diagnosticassays for such diseases or conditions.

In another aspect, the invention provides methods for treating a diseaseor abnormal condition by administering to a patient in need of suchtreatment a substance that modulates the activity or expression of apolypeptide having sequences selected from the group consisting of SEQID NO:4 to SEQ ID NO:6.

Agents that modulate (i.e., increase, decrease, or block) MMP activityor expression may be identified by incubating a putative modulator witha cell containing an MMP polypeptide or polynucleotide and determiningthe effect of the putative modulator on MMP activity or expression. Theselectivity of a compound that modulates the activity of MMP can beevaluated by comparing its effects on MMP to its effect on other MMPcompounds. Following identification of compounds that modulate MMPactivity or expression, such compounds may be further tested in otherassays including, but not limited to, in vivo models, in order toconfirm or quantitate their activity. Selective modulators may include,for example, antibodies and other proteins, peptides, or organicmolecules that specifically bind to an MMP polypeptide or a MMP-encodingnucleic acid. Modulators of MMP activity will be therapeutically usefulin treatment of diseases and physiological conditions in which normal oraberrant MMP activity is involved. MMP polynucleotides, polypeptides,and modulators may be used in the treatment of such diseases andconditions as metabolic diseases and disorders (e.g., type 2 diabetes,obesity, cardiovascular, dyslipidemias, adipogenesis, retinopathies,neuropathies, nephropathies etc.), proliferative diseases and cancers(e.g., different cancers such as breast, colon, lung, etc., tumorgrowth, tumor invasion, and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc.), hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.), CNS disorders (e.g., degenerative disorders such as Parkinson's,Alzheimer's, etc.), inflammatory conditions (e.g., Chron's disease,arthritis), diseases related to cell differentiation and homeostasis,cardiomyopathy, atherosclerosis, thromboembolic diseases, Sjögren'ssyndrome, renal failure, periodontal diseases, retinalneovascularization, wound healing, and neurodegenerative diseasesincluding, for example, Alzheimer's disease, multiple sclerosis,Parkinson's disease, and motoneuron disease, among others.

Methods of the invention to identify modulators include variations onany of the methods described above to identify binding partnercompounds, the variations including techniques wherein a binding partnercompound has been identified and the binding assay is carried out in thepresence and absence of a candidate modulator. A modulator is identifiedin those instances where binding between the MMP polypeptide and thebinding partner compound changes in the presence of the candidatemodulator compared to binding in the absence of the candidate modulatorcompound. A modulator that increases binding between the MMP polypeptideand the binding partner compound is described as an enhancer oractivator, and a modulator that decreases binding between the MMPpolypeptide and the binding partner compound is described as aninhibitor. Following identification of modulators, such compounds may befurther tested in other assays including, but not limited to, in vivomodels, in order to confirm or quantitate their activity as modulators.

The invention also comprehends high-throughput screening (HTS) assays toidentify compounds that interact with or inhibit biological activity(i.e., affect enzymatic activity, binding activity, etc.) of a MMPpolypeptide. HTS assays permit screening of large numbers of compoundsin an efficient manner. Cell-based HTS systems are contemplated toinvestigate MMP-ligand interaction. HTS assays are designed to identify“hits” or “lead compounds” having the desired property, from whichmodifications can be designed to improve the desired property. Chemicalmodification of the “hit” or “lead compound” is often based on anidentifiable structure/activity relationship between the “hit” and theMMP polypeptide.

Another aspect of the present invention is directed to methods ofidentifying compounds which modulate (i.e., increase or decrease) anactivity of MMP comprising contacting MMP with a compound, anddetermining whether the compound modifies activity of MMP. The activityin the presence of the test compared is measured to the activity in theabsence of the test compound. Where the activity of the samplecontaining the test compound is higher than the activity in the samplelacking the test compound, the compound will have increased activity.Similarly, where the activity of the sample containing the test compoundis lower than the activity in the sample lacking the test compound, thecompound will have inhibited activity. Following the identification ofcompounds that modulate an activity of MMP, such compounds can befurther tested in other assays including, but not limited to, in vivomodels, in order to confirm or quantitate their activity.

The present invention is particularly useful for screening compounds byusing MMP in any of a variety of drug screening techniques. Thecompounds to be screened include (which may include compounds which aresuspected to modulate MMP activity), but are not limited to,extracellular, intracellular, biologic or chemical origin. The NMPpolypeptide employed in such a test may be in any form, preferably, freein solution, attached to a solid support, borne on a cell surface orlocated intracellularly. One skilled in the art can, for example,measure the formation of complexes between MMP and the compound beingtested. Alternatively, one skilled in the art can examine the diminutionin complex formation between MMP and its substrate caused by thecompound being tested.

The activity of MMP polypeptides of the invention can be determined by,for example, examining the ability to bind or be activated byappropriate ligands, such as low molecular weight steroids and fattyacids. The activity of the MMPs can be assayed by, for example,competition-binding assays and coactivator-interaction assays (see,e.g., Makishima et al, 1999, Science, 284, 1362-1365; Parks et al.,Science, 284, 1365-1367). Alternatively, the activity of MMPpolypeptides can be assayed by examining their ability to cleave a knownMMP substrate.

The modulators of the invention exhibit a variety of chemicalstructures, which can be generally grouped into non-peptide mimetics ofnatural NMP ligands, peptide and non-peptide allosteric effectors ofMMPs, and peptides that may function as activators or inhibitors(competitive, uncompetitive and non-competitive) (e.g., antibodyproducts) of MMPs. The invention does not restrict the sources forsuitable modulators, which may be obtained from natural sources such asplant, animal or mineral extracts, or non-natural sources such as smallmolecule libraries, including the products of combinatorial chemicalapproaches to library construction, and peptide libraries.

Other assays can be used to examine enzymatic activity including, butnot limited to, photometric, radiometric, HPLC, electrochemical, and thelike, which are described in, for example, Enzyme Assays: A PracticalApproach, eds. R. Eisenthal and M. J. Danson, 1992, Oxford UniversityPress, which is incorporated herein by reference in its entirety.

The use of cDNAs encoding MMPs in drug discovery programs is well-known;assays capable of testing thousands of unknown compounds per day inhigh-throughput screens (HTSs) are thoroughly documented. The literatureis replete with examples of the use of radiolabeled ligands in HTSbinding assays for drug discovery (see Williams, Medicinal ResearchReviews, 1991, 11, 147-184; Sweetnam, et al., J. Natural Products, 1993,56, 441-455 for review).

A variety of heterologous systems is available for functional expressionof recombinant polypeptides that are well known to those skilled in theart. Such systems include bacteria (Strosberg, et al., Trends inPharmacological Sciences, 1992, 13, 95-98), yeast (Pausch, Trends inBiotechnology, 1997, 15, 487-494), several kinds of insect cells (VandenBroeck, Int. Rev. Cytology, 1996, 164, 189-268), amphibian cells(Jayawickreme et al., Current Opinion in Biotechnology, 1997, 8,629-634) and several mammalian cell lines (CHO, HEK-293, COS, etc.; seeGerhardt, et al., Eur. J. Pharmacology, 1997, 334, 1-23). These examplesdo not preclude the use of other possible cell expression systems,including cell lines obtained from nematodes (PCT application WO98/37177).

In preferred embodiments of the invention, methods of screening forcompounds that modulate MMP activity comprise contacting test compoundswith MMP and assaying for the presence of a complex between the compoundand MMP. In such assays, the ligand is typically labeled. After suitableincubation, free ligand is separated from that present in bound form,and the amount of free or uncomplexed label is a measure of the abilityof the particular compound to bind to MMP.

The invention contemplates a multitude of assays to screen and identifyinhibitors of ligand binding to MMPs. In one example, the MMP isimmobilized and interaction with a binding partner is assessed in thepresence and absence of a candidate modulator such as an inhibitorcompound. In another example, interaction between the MMP and itsbinding partner is assessed in a solution assay, both in the presenceand absence of a candidate inhibitor compound. In either assay, aninhibitor is identified as a compound that decreases binding between theMMP and its binding partner. Following the identification of compoundswhich inhibit ligand binding to MMP, such compounds may be furthertested in other assays including, but not limited to, in vivo models, inorder to confirm or quantitate their activity. Another contemplatedassay involves a variation of the dihybrid assay wherein an inhibitor ofprotein/protein interactions is identified by detection of a positivesignal in a transformed or transfected host cell, as described in PCTpublication number WO 95/20652, published Aug. 3, 1995.

Candidate modulators contemplated by the invention include compoundsselected from libraries of either potential activators or potentialinhibitors. There are a number of different libraries used for theidentification of small molecule modulators, including: (1) chemicallibraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides or organicmolecules. Chemical libraries consist of random chemical structures,some of which are analogs of known compounds or analogs of compoundsthat have been identified as “hits” or “leads” in other drug discoveryscreens, some of which are derived from natural products, and some ofwhich arise from non-directed synthetic organic chemistry. Naturalproduct libraries are collections of microorganisms, animals, plants, ormarine organisms which are used to create mixtures for screening by: (1)fermentation and extraction of broths from soil, plant or marinemicroorganisms or (2) extraction of plants or marine organisms. Naturalproduct libraries include polyketides, non-ribosomal peptides, andvariants (non-naturally occurring) thereof. For a review, see Science282:63-68 (1998). Combinatorial libraries are composed of large numbersof peptides, oligonucleotides, or organic compounds as a mixture. Theselibraries are relatively easy to prepare by traditional automatedsynthesis methods, PCR, cloning, or proprietary synthetic methods. Ofparticular interest are non-peptide combinatorial libraries. Still otherlibraries of interest include peptide, protein, peptidomimetic,multiparallel synthetic collection, recombinatorial, and polypeptidelibraries. For a review of combinatorial chemistry and libraries createdtherefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).Identification of modulators through use of the various librariesdescribed herein permits modification of the candidate “hit” (or “lead”)to optimize the capacity of the “hit” to modulate activity.

Still other candidate inhibitors contemplated by the invention can bedesigned and include soluble forms of binding partners, as well as suchbinding partners as chimeric, or fusion, proteins. A “binding partner”as used herein broadly encompasses non-peptide modulators, as well assuch peptide modulators as neuropeptides other than natural ligands,antibodies, antibody fragments, and modified compounds comprisingantibody domains that are immunospecific for the expression product ofthe identified MMP gene.

The polypeptides of the invention are employed as a research tool foridentification, characterization and purification of interacting,regulatory proteins. Appropriate labels are incorporated into thepolypeptides of the invention by various methods known in the art andthe polypeptides are used to capture interacting molecules. For example,molecules are incubated with the labeled polypeptides, washed to removeunbound polypeptides, and the polypeptide complex is quantified. Dataobtained using different concentrations of polypeptide are used tocalculate values for the number, affinity, and association ofpolypeptide with the protein complex.

Labeled polypeptides are also useful as reagents for the purification ofmolecules with which the polypeptide interacts including, but notlimited to, inhibitors. In one embodiment of affinity purification, apolypeptide is covalently coupled to a chromatography column. Cells andtheir membranes are extracted, and various cellular subcomponents arepassed over the column. Molecules bind to the column by virtue of theiraffinity to the polypeptide. The polypeptide-complex is recovered fromthe column, dissociated and the recovered molecule is subjected toprotein sequencing. This amino acid sequence is then used to identifythe captured molecule or to design degenerate oligonucleotides forcloning the corresponding gene from an appropriate cDNA library.

Alternatively, compounds may be identified which exhibit similarproperties to the ligand for the MMP of the invention, but which aresmaller and exhibit a longer half time than the endogenous ligand in ahuman or animal body. When an organic compound is designed, a moleculeaccording to the invention is used as a “lead” compound. The design ofmimetics to known pharmaceutically active compounds is a well-knownapproach in the development of pharmaceuticals based on such “lead”compounds. Mimetic design, synthesis and testing are generally used toavoid randomly screening a large number of molecules for a targetproperty. Furthermore, structural data deriving from the analysis of thededuced amino acid sequences encoded by the DNAs of the presentinvention are useful to design new drugs, more specific and thereforewith a higher pharmacological potency.

Comparison of the protein sequences of the present invention with thesequences present in all the available databases showed homology withthe known MMPs. Accordingly, computer modeling can be used to develop aputative tertiary structure of the proteins of the invention based onthe available information of the conserved domains of the polypeptidesof the present invention. Thus, novel ligands based on the predictedstructure of MMP can be designed.

In a particular embodiment, the novel molecules identified by thescreening methods according to the invention are low molecular weightorganic molecules, in which case a composition or pharmaceuticalcomposition can be prepared thereof for oral intake, such as in tablets.The compositions, or pharmaceutical compositions, comprising the nucleicacid molecules, vectors, polypeptides, antibodies and compoundsidentified by the screening methods described herein, can be preparedfor any route of administration including, but not limited to, oral,intravenous, cutaneous, subcutaneous, nasal, intramuscular orintraperitoneal. The nature of the carrier or other ingredients willdepend on the specific route of administration and particular embodimentof the invention to be administered. Examples of techniques andprotocols that are useful in this context are, inter alia, found inRemington's Pharmaceutical Sciences, 16^(th) edition, Osol, A (ed.),1980, which is incorporated herein by reference in its entirety.

The dosage of these low molecular weight compounds will depend on thedisease state or condition to be treated and other clinical factors suchas weight and condition of the human or animal and the route ofadministration of the compound. For treating human or animals, betweenapproximately 0.5 mg/kg of body weight to 500 mg/kg of body weight ofthe compound can be administered. Therapy is typically administered atlower dosages and is continued until the desired therapeutic outcome isobserved.

The present compounds and methods, including nucleic acid molecules,polypeptides, antibodies, compounds identified by the screening methodsdescribed herein, have a variety of pharmaceutical applications and maybe used, for example, to treat or prevent unregulated cellular growth,such as cancer cell and tumor growth. In a particular embodiment, thepresent molecules are used in gene therapy. For a review of gene therapyprocedures, see e.g. Anderson, Science, 1992, 256, 808-813, which isincorporated herein by reference in its entirety.

The present invention also encompasses a method of agonizing(stimulating) or antagonizing a MMP natural binding partner associatedactivity in a mammal comprising administering to said mammal an agonistor antagonist to one of the above disclosed polypeptides in an amountsufficient to effect said agonism or antagonism. One embodiment of thepresent invention, then, is a method of treating diseases in a mammalwith an agonist or antagonist of the protein of the present inventioncomprises administering the agonist or antagonist to a mammal in anamount sufficient to agonize or antagonize MMP-associated functions.

Exemplary diseases and conditions amenable to treatment based on thepresent invention include, but are not limited to, metabolic diseasesand disorders (e.g., type 2 diabetes, obesity, cardiovascular,dyslipidemias, adipogenesis, retinopathies, neuropathies, nephropathiesetc.), proliferative diseases and cancers (e.g., different cancers suchas breast, colon, lung, etc., tumor growth, tumor invasion, andhyperproliferative disorders such as psoriasis, prostate hyperplasia,etc.), hormonal disorders (e.g., male/female hormonal replacement,polycystic ovarian syndrome, alopecia, etc.), CNS disorders (e.g.,degenerative disorders such as Parkinson's, Alzheimer's, etc.),inflammatory conditions (e.g., Chron's disease, arthritis), diseasesrelated to cell differentiation and homeostasis, cardiomyopathy,atherosclerosis, thromboembolic diseases, Sjögren's syndrome, renalfailure, periodontal diseases, retinal neovascularization, woundhealing, and neurodegenerative diseases including, for example,Alzheimer's disease, multiple sclerosis, Parkinson's disease, andmotoneuron disease, among others.

Methods of determining the dosages of compounds to be administered to apatient and modes of administering compounds to an organism aredisclosed in U.S. application Ser. No. 08/702,282, filed Aug. 23, 1996and International patent publication number WO 96/22976, published Aug.1, 1996, both of which are incorporated herein by reference in theirentirety, including any drawings, figures or tables. Those skilled inthe art will appreciate that such descriptions are applicable to thepresent invention and can be easily adapted to it.

The proper dosage depends on various factors such as the type of diseasebeing treated, the particular composition being used and the size andphysiological condition of the patient. Therapeutically effective dosesfor the compounds described herein can be estimated initially from cellculture and animal models. For example, a dose can be formulated inanimal models to achieve a circulating concentration range thatinitially takes into account the IC₅₀ as determined in cell cultureassays. The animal model data can be used to more accurately determineuseful doses in humans.

Plasma half-life and bio-distribution of the drug and metabolites in theplasma, tumors and major organs can also be determined to facilitate theselection of drugs most appropriate to inhibit a disorder. Suchmeasurements can be carried out. For example, HPLC analysis can beperformed on the plasma of animals treated with the drug and thelocation of radiolabeled compounds can be determined using detectionmethods such as X-ray, CAT scan and MRI. Compounds that show potentinhibitory activity in the screening assays, but have poorpharmacokinetic characteristics, can be optimized by altering thechemical structure and retesting. In this regard, compounds displayinggood pharmacokinetic characteristics can be used as a model.

Toxicity studies can also be carried out by measuring the blood cellcomposition. For example, toxicity studies can be carried out in asuitable animal model as follows: 1) the compound is administered tomice (an untreated control mouse should also be used); 2) blood samplesare periodically obtained via the tail vein from one mouse in eachtreatment group; and 3) the samples are analyzed for red and white bloodcell counts, blood cell composition and the percent of lymphocytesversus polymorphonuclear cells. A comparison of results for each dosingregime with the controls indicates if toxicity is present.

At the termination of each toxicity study, further studies can becarried out by sacrificing the animals (preferably, in accordance withthe American Veterinary Medical Association guidelines Report of theAmerican Veterinary Medical Assoc. Panel on Euthanasia, Journal ofAmerican Veterinary Medical Assoc., 202:229-249, 1993). Representativeanimals from each treatment group can then be examined by gross necropsyfor immediate evidence of metastasis, unusual illness or toxicity. Grossabnormalities in tissue are noted and tissues are examinedhistologically. Compounds causing a reduction in body weight or bloodcomponents are less preferred, as are compounds having an adverse effecton major organs. In general, the greater the adverse effect the lesspreferred the compound.

For the treatment of many diseases, the expected daily dose of ahydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably1 to 250 mg/day, and most preferably 1 to 50 mg/day. Drugs can bedelivered less frequently provided plasma levels of the active moietyare sufficient to maintain therapeutic effectiveness. Plasma levelsshould reflect the potency of the drug. Generally, the more potent thecompound the lower the plasma levels necessary to achieve efficacy.

As discussed above, it is well known that MMPs are expressed in manydifferent tissues and regions, including in the brain. MMP mRNAtranscripts may found in many other tissues, including, but not limitedto pancreas (and particularly pancreatic islet tissue), pituitary,skeletal muscle, adipose tissue, liver, and thyroid, and may be found inmany other tissues.

Sequences selected from the group consisting of SEQ ID NO:1 to SEQ IDNO:3 will, as detailed above, enable screening the endogenoushormones/ligands which activate, agonize, or antagonize MMP and forcompounds with potential utility in treating disorders including, butnot limited to, metabolic diseases and disorders (e.g., type 2 diabetes,obesity, cardiovascular, dyslipidemias, adipogenesis, retinopathies,neuropathies, nephropathies etc.), proliferative diseases and cancers(e.g., different cancers such as breast, colon, lung, etc., tumorgrowth, tumor invasion, and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc.), hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.), CNS disorders (e.g., degenerative disorders such as Parkinson's,Alzheimer's, etc.), inflammatory conditions (e.g., Chron's disease,arthritis), diseases related to cell differentiation and homeostasis,cardiomyopathy, atherosclerosis, thromboembolic diseases, Sjögren'ssyndrome, renal failure, periodontal diseases, retinalneovascularization, wound healing, and neurodegenerative diseasesincluding, for example, Alzheimer's disease, multiple sclerosis,Parkinson's disease, and motoneuron disease, among others.

The attached Sequence Listing contains the sequences of thepolynucleotides and polypeptides of the invention and is incorporatedherein by reference in its entirety.

Methods of Screening Human Subjects

Thus in yet another embodiment, the invention provides genetic screeningprocedures that entail analyzing a person's genome—in particular theiralleles for the MMPs of the invention—to determine whether theindividual possesses a genetic characteristic found in other individualsthat are considered to be afflicted with, or at risk for, developing amental disorder or disease of the brain that is suspected of having ahereditary component. For example, in one embodiment, the inventionprovides a method for determining a potential for developing a disorderaffecting the brain in a human subject comprising the steps of analyzingthe coding sequence of one or more MMP genes from the human subject; anddetermining development potential for the disorder in said human subjectfrom the analyzing step.

More particularly, the invention provides a method of screening a humansubject to diagnose a disorder affecting the brain or geneticpredisposition therefor, comprising the steps of: (a) assaying nucleicacid of a human subject to determine a presence or an absence of amutation altering the amino acid sequence, expression, or biologicalactivity of at least one MMP that is expressed in the brain, wherein theMMP comprises an amino acid sequence selected from the group consistingof SEQ ID NO:1 to SEQ ID NO:3, or an allelic variant thereof, andwherein the nucleic acid corresponds to the gene encoding the MMP; and(b) diagnosing the disorder or predisposition from the presence orabsence of said mutation, wherein the presence of a mutation alteringthe amino acid sequence, expression, or biological activity of allele inthe nucleic acid correlates with an increased risk of developing thedisorder.

By “human subject” is meant any human being, human embryo, or humanfetus. It will be apparent that methods of the present invention will beof particular interest to individuals that have themselves beendiagnosed with a disorder affecting the brain or have relatives thathave been diagnosed with a disorder affecting the brain.

By “screening for an increased risk” is meant determination of whether agenetic variation exists in the human subject that correlates with agreater likelihood of developing a disorder affecting the brain thanexists for the human population as a whole, or for a relevant racial orethnic human sub-population to which the individual belongs. Bothpositive and negative determinations (i.e., determinations that agenetic predisposition marker is present or is absent) are intended tofall within the scope of screening methods of the invention. Inpreferred embodiments, the presence of a mutation altering the sequenceor expression of at least one MMP allele in the nucleic acid iscorrelated with an increased risk of developing mental disorder, whereasthe absence of such a mutation is reported as a negative determination.

The “assaying” step of the invention may involve any techniquesavailable for analyzing nucleic acid to determine its characteristics,including but not limited to well-known techniques such as single-strandconformation polymorphism analysis (SSCP) [Orita et al., Proc Natl.Acad. Sci. USA, 86: 2766-2770 (1989)]; heteroduplex analysis [White etal., Genomics, 12: 301-306 (1992)]; denaturing gradient gelelectrophoresis analysis [Fischer et al., Proc. Natl. Acad. Sci. USA,80: 1579-1583 (1983); and Riesner et al., Electrophoresis, 10: 377-389(1989)]; DNA sequencing; RNase cleavage [Myers et al., Science, 230:1242-1246 (1985)]; chemical cleavage of mismatch techniques [Rowley etal., Genomics, 30: 574-582 (1995); and Roberts et al., Nucl. Acids Res.,25: 3377-3378 (1997)]; restriction fragment length polymorphismanalysis; single nucleotide primer extension analysis [Shumaker et al.,Hum. Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7:606-614 (1997)]; 5′ nuclease assays [Pease et al., Proc. Natl. Acad.Sci. USA, 91:5022-5026 (1994)]; DNA Microchip analysis [Ramsay, G.,Nature Biotechnology, 16: 40-48 (1999); and Chee et al., U.S. Pat. No.5,837,832]; and ligase chain reaction [Whiteley et al., U.S. Pat. No.5,521,065]. [See generally, Schafer and Hawkins, Nature Biotechnology,16: 33-39 (1998).] All of the foregoing documents are herebyincorporated by reference in their entirety.

Thus, in one preferred embodiment involving screening MMP sequences, forexample, the assaying step comprises at least one procedure selectedfrom the group consisting of: (a) determining a nucleotide sequence ofat least one codon of at least one MMP allele of the human subject; (b)performing a hybridization assay to determine whether nucleic acid fromthe human subject has a nucleotide sequence identical to or differentfrom one or more reference sequences; (c) performing a polynucleotidemigration assay to determine whether nucleic acid from the human subjecthas a nucleotide sequence identical to or different from one or morereference sequences; and (d) performing a restriction endonucleasedigestion to determine whether nucleic acid from the human subject has anucleotide sequence identical to or different from one or more referencesequences.

In a highly preferred embodiment, the assaying involves sequencing ofnucleic acid to determine nucleotide sequence thereof, using anyavailable sequencing technique. [See, e.g., Sanger et al., Proc. Natl.Acad. Sci. (USA), 74: 5463-5467 (1977) (dideoxy chain terminationmethod); Mirzabekov, TIBTECH, 12: 27-32 (1994) (sequencing byhybridization); Drmanac et al., Nature Biotechnology, 16: 54-58 (1998);U.S. Pat. No. 5,202,231; and Science, 260: 1649-1652 (1993) (sequencingby hybridization); Kieleczawa et al., Science, 258: 1787-1791 (1992)(sequencing by primer walking); (Douglas et al., Biotechniques, 14:824-828 (1993) (Direct sequencing of PCR products); and Akane et al.,Biotechniques 16: 238-241 (1994); Maxam and Gilbert, Meth. Enzymol., 65:499-560 (1977) (chemical termination sequencing), all incorporatedherein by reference.] The analysis may entail sequencing of the entireMMP gene genomic DNA sequence, or portions thereof; or sequencing of theentire MMP coding sequence or portions thereof. In some circumstances,the analysis may involve a determination of whether an individualpossesses a particular allelic variant, in which case sequencing of onlya small portion of nucleic acid—enough to determine the sequence of aparticular codon characterizing the allelic variant—is sufficient. Thisapproach is appropriate, for example, when assaying to determine whetherone family member inherited the same allelic variant that has beenpreviously characterized for another family member, or, more generally,whether a person's genome contains an allelic variant that has beenpreviously characterized and correlated with a mental disorder having aheritable component.

In another highly preferred embodiment, the assaying step comprisesperforming a hybridization assay to determine whether nucleic acid fromthe human subject has a nucleotide sequence identical to or differentfrom one or more reference sequences. In a preferred embodiment, thehybridization involves a determination of whether nucleic acid derivedfrom the human subject will hybridize with one or more oligonucleotides,wherein the oligonucleotides have nucleotide sequences that correspondidentically to a portion of the MMP gene sequence taught herein, or thatcorrespond identically except for one mismatch. The hybridizationconditions are selected to differentiate between perfect sequencecomplementarity and imperfect matches differing by one or more bases.Such hybridization experiments thereby can provide single nucleotidepolymorphism sequence information about the nucleic acid from the humansubject, by virtue of knowing the sequences of the oligonucleotides usedin the experiments.

Several of the techniques outlined above involve an analysis wherein oneperforms a polynucleotide migration assay, e.g., on a polyacrylamideelectrophoresis gel (or in a capillary electrophoresis system), underdenaturing or non-denaturing conditions. Nucleic acid derived from thehuman subject is subjected to gel electrophoresis, usually adjacent to(or co-loaded with) one or more reference nucleic acids, such asreference MMP encoding sequences having a coding sequence identical toall or a portion of SEQ ID NOS: 1 to 3 (or identical except for oneknown polymorphism). The nucleic acid from the human subject and thereference sequence(s) are subjected to similar chemical or enzymatictreatments and then electrophoresed under conditions whereby thepolynucleotides will show a differential migration pattern, unless theycontain identical sequences. [See generally Ausubel et al. (eds.),Current Protocols in Molecular Biology, New York: John Wiley & Sons,Inc. (1987-1999); and Sambrook et al., (eds.), Molecular Cloning, ALaboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1989), both incorporated herein by reference in theirentirety.]

In the context of assaying, the term “nucleic acid of a human subject”is intended to include nucleic acid obtained directly from the humansubject (e.g., DNA or RNA obtained from a biological sample such as ablood, tissue, or other cell or fluid sample); and also nucleic acidderived from nucleic acid obtained directly from the human subject. Byway of non-limiting examples, well known procedures exist for creatingcDNA that is complementary to RNA derived from a biological sample froma human subject, and for amplifying (e.g., via polymerase chain reaction(PCR)) DNA or RNA derived from a biological sample obtained from a humansubject. Any such derived polynucleotide which retains relevantnucleotide sequence information of the human subject's own DNA/RNA isintended to fall within the definition of “nucleic acid of a humansubject” for the purposes of the present invention.

In the context of assaying, the term “mutation” includes addition,deletion, and/or substitution of one or more nucleotides in the MMP genesequence (e.g., as compared to the MMP-encoding sequences set forth ofSEQ ID NO:1 to SEQ ID NO:3, and other polymorphisms that occur inintrons (where introns exist) and that are identifiable via sequencing,restriction fragment length polymorphism, or other techniques. Thevarious activity examples provided herein permit determination ofwhether a mutation modulates activity of the relevant MMP in thepresence or absence of various test substances.

In a related embodiment, the invention provides methods of screening aperson's genotype with respect to the MMP of the invention, andcorrelating such genotypes with diagnoses for disease or withpredisposition for disease (for genetic counseling). For example, theinvention provides a method of screening for an MMP hereditary mentaldisorder genotype in a human patient, comprising the steps of: (a)providing a biological sample comprising nucleic acid from the patient,the nucleic acid including sequences corresponding to said patient's MMPalleles; (b) analyzing the nucleic acid for the presence of a mutationor mutations; (c) determining a MMP genotype from the analyzing step;and (d) correlating the presence of a mutation in an MMP allele with ahereditary mental disorder genotype. In a preferred embodiment, thebiological sample is a cell sample containing human cells that containgenomic DNA of the human subject. The analyzing can be performedanalogously to the assaying described in preceding paragraphs. Forexample, the analyzing comprises sequencing a portion of the nucleicacid (e.g., DNA or RNA), the portion comprising at least one codon ofthe MMP alleles.

Although more time consuming and expensive than methods involvingnucleic acid analysis, the invention also may be practiced by assayingone or more proteins of a human subject to determine the presence orabsence of an amino acid sequence variation in MMP protein from thehuman subject. Such protein analyses may be performed, e.g., byfragmenting MMP protein via chemical or enzymatic methods and sequencingthe resultant peptides; or by Western analyses using an antibody havingspecificity for a particular allelic variant of the MMP.

The invention also provides materials that are useful for performingmethods of the invention. For example, the present invention providesoligonucleotides useful as probes in the many analyzing techniquesdescribed above. In general, such oligonucleotide probes comprise 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 nucleotides that have a sequence that isidentical, or exactly complementary, to a portion of a human MMP genesequence taught herein (or allelic variant thereof), or that isidentical or exactly complementary except for one nucleotidesubstitution. In a preferred embodiment, the oligonucleotides have asequence that corresponds in the foregoing manner to a human MMP codingsequence taught herein, and in particular, the coding sequences setforth in SEQ ID NO:1 to SEQ ID NO:3. In one variation, anoligonucleotide probe of the invention is purified and isolated. Inanother variation, the oligonucleotide probe is labeled, e.g., with aradioisotope, chromophore, or fluorophore. In yet another variation, theprobe is covalently attached to a solid support. [See generally Ausubelet al. and Sambrook et al., supra.]

In a related embodiment, the invention provides kits comprising reagentsthat are useful for practicing methods of the invention. For example,the invention provides a kit for screening a human subject to diagnose amental disorder or a genetic predisposition therefor, comprising, inassociation: (a) an oligonucleotide useful as a probe for identifyingpolymorphisms in a human MMP gene, the oligonucleotide comprising 6-50nucleotides that have a sequence that is identical or exactlycomplementary to a portion of a human MMP gene sequence or MMP codingsequence, except for one sequence difference selected from the groupconsisting of a nucleotide addition, a nucleotide deletion, ornucleotide substitution; and (b) a media packaged with theoligonucleotide containing information identifying polymorphismsidentifiable with the probe that correlate with mental disorder or agenetic predisposition therefor. Exemplary information-containing mediainclude printed paper package inserts or packaging labels; and magneticand optical storage media that are readable by computers or machinesused by practitioners who perform genetic screening and counselingservices. The practitioner uses the information provided in the media tocorrelate the results of the analysis with the oligonucleotide with adiagnosis. In a preferred variation, the oligonucleotide is labeled.

In still another embodiment, the invention provides methods ofidentifying those allelic variants of MMP of the invention thatcorrelate with mental disorders. For example, the invention provides amethod of identifying an MMP allelic variant that correlates with amental disorder, comprising steps of: (a) providing a biological samplecomprising nucleic acid from a human patient diagnosed with a mentaldisorder, or from the patient's genetic progenitors or progeny; (b)analyzing the nucleic acid for the presence of a mutation or mutationsin at least one MMP that is expressed in the brain, wherein the at leastone MMP comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:3 or an allelic variant thereof,and wherein the nucleic acid includes sequence corresponding to the geneor genes encoding the at least one MMP; (c) determining a genotype forthe patient for the at least one MMP from said analyzing step; and (d)identifying an allelic variant that correlates with the mental disorderfrom the determining step. To expedite this process, it may be desirableto perform linkage studies in the patients (and possibly their families)to correlate chromosomal markers with disease states. The chromosomallocalization data provided herein facilitates identifying an involvedMMP with a chromosomal marker.

The foregoing method can be performed to correlate the MMP of theinvention to a number of disorders having hereditary components that arecausative or that predispose persons to the disorder. For example, inone preferred variation, the disorder is a mental disorder.

Also contemplated as part of the invention are polynucleotides thatcomprise the allelic variant sequences identified by such methods, andpolypeptides encoded by the allelic variant sequences, andoligonucleotide and oligopeptide fragments thereof that embody themutations that have been identified. Such materials are useful in invitro cell-free and cell-based assays for identifying lead compounds andtherapeutics for treatment of the disorders. For example, the variantsare used in activity assays, binding assays, and assays to screen foractivity modulators described herein. In one preferred embodiment, theinvention provides a purified and isolated polynucleotide comprising anucleotide sequence encoding a MMP allelic variant identified accordingto the methods described above; and an oligonucleotide that comprisesthe sequences that differentiate the allelic variant from the MMPsequences set forth in SEQ ID NO:1 to SEQ ID NO:3. The invention alsoprovides a vector comprising the polynucleotide (preferably anexpression vector); and a host cell transformed or transfected with thepolynucleotide or vector. The invention also provides an isolated cellline that is expressing the allelic variant NMP polypeptide; purifiedcell membranes from such cells; purified polypeptide; and syntheticpeptides that embody the allelic variation amino acid sequence. In oneparticular embodiment, the invention provides a purified polynucleotidecomprising a nucleotide sequence encoding an MMP protein of a human thatis affected with a mental disorder; wherein said polynucleotidehybridizes to the complement of a sequence selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:3 under the followinghybridization conditions: (a) hybridization for 16 hours at 42° C. in ahybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10%dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in awash solution comprising 0.1×SSC and 1% SDS; and wherein thepolynucleotide encodes an MM amino acid sequence that differs from asequence selected from the group consisting of SEQ ID NO:4 to SEQ IDNO:6, by at least one residue.

An exemplary assay for using the allelic variants is a method foridentifying a modulator of MMP biological activity, comprising the stepsof: (a) contacting a cell expressing the allelic variant in the presenceand in the absence of a putative modulator compound; (b) measuring MMPbiological activity in the cell; and (c) identifying a putativemodulator compound in view of decreased or increased MMP biologicalactivity in the presence versus absence of the putative modulator.

Additional features of the invention will be apparent from the followingExamples. Examples 1 is actual while the remaining Examples areprophetic. Additional features and variations of the invention will beapparent to those skilled in the art from the entirety of thisapplication, including the detailed description, and all such featuresare intended as aspects of the invention. Likewise, features of theinvention described herein can be re-combined into additionalembodiments that also are intended as aspects of the invention,irrespective of whether the combination of features is specificallymentioned above as an aspect or embodiment of the invention. Also, onlysuch limitations which are described herein as critical to the inventionshould be viewed as such; variations of the invention lackinglimitations which have not been described herein as critical areintended as aspects of the invention.

EXAMPLE 1

Identification of MMP

A. Database Search

The Incyte LifeSeq databases LGTemplatesAUG1999, LGTemplatesOCT1999, andLGTemplatesDEC1999, and the Celera database Releases 1.04-1.05 weresearched using BLAST and nucleotide/protein sequence from known MMPs. Acollection of query amino acid sequences derived from MMPs was used tosearch the DNA sequence databases using TBLASTN and alignments with anE-value lower than 10⁻⁵ were collected from each BLAST search. The newsequences found were then BLAST searched against proprietary databasesin order to eliminate known MMPs and identify novel MMPs.

Briefly, the BLAST algorithm (Basic Local Alignment Search Tool) issuitable for determining sequence similarity (Altschul et al., J. Mol.Biol., 1990, 215, 403-410, which is incorporated herein by reference inits entirety). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to find HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Extension for the word hits in each direction are haltedwhen: 1) the cumulative alignment score falls off by the quantity X fromits maximum achieved value; 2) the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or 3) the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad.Sci. USA, 1992, 89, 10915-10919, which is incorporated herein byreference in its entirety) alignments (B) of 50, expectation (E) of 10,M=5, N=4, and a comparison of both strands.

The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993,90, 5873-5787, which is incorporated herein by reference in itsentirety) and Gapped BLAST perform a statistical analysis of thesimilarity between two sequences. One measure of similarity provided bythe BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a MMP gene or cDNA if the smallestsum probability in comparison of the test nucleic acid to an MMP nucleicacid is less than about 1 to about 10⁻⁵.

The following Table 5 contains the sequences of the polynucleotides andpolypeptides of the invention. The coding regions within thepolynucleotide sequences are identified by underlining.

TABLE 5 The following DNA sequence MMPU1 <SEQ ID NO. 1> was identifiedin H. sapiens.GCTTCAGCTGAAGAAAGAGAGGAATGAAGCGCCTTCTGCTTCTGTGTTTGTTCTTTATAACATTTTCTTCTGCATTTCCCTTAGTCCGGATGACGGAAAATGAAGAAAATATGCAACTGGCTCAGGCATATCTCAACCAGTTCTACTCTCTTGAAATAGAAGCGAATCATCTTGTTCAAAGCAAGAATAGGAGTCTCATAGATGACAAAATTCGGGAAATGCAAGCATTTTTTGGATTGACAGTGACTGGAAAACTGGACTCAAACACCCTTGAGATCATGAAGACACCCAGGTGTGGGGTGCCTGATGTGGGCCAGTATGGCTACACCCTCCCTGGGTGGAGAAAATACAACCTCACCTACAGAATAATAAACTATACTCCGGATATGGGACGAGCTGCTGTGGATGAGGCTATCCAAGAAGGTTTAGAAGTGTGGAGCAAAGTCACTCCACTAAAATTCACCAAGATTTCAAAGGGGATTGCAGACATCATGATTGCCTTTAGGACTCGAGTCCATGGTCCGTGTCCTCGCTATTTTGATGGTCCCTTCGGAGTGCTTGGCCATGCCTTTCCTCCTGGTCCGGCTCTGGGTGGTGACACTCATTTTGATGAGGATGAAAACTGGACCAAGGATGGAGCAGGATTCAACTTGTTTCTTGTGGCTGCTCATGAATTTGGTCATGCACTGGGGCTCTCTCACTCCAATGATCAAACAGCCTTGATGTTCCCAAATTATGTCTCCCTGGATCCCAGAAAATACCCACTTTCTCAGGATGATATCAATGGAATCCAGTCCATCTATGGAGGTCTGCCTAAGGTACCTGCTAAGCCAAAGGAACCCACTATACCCCATGCCTGTGACCCTGACTTGACTTTTGACGCTATCACAACTTTCCGCAGAGAAGTAATGTTCTTTAAAGGCAGGCACCTATGGAGGATCTATTATGATATCACGGATGTTGAGTTTGAATTAATTGCTTCATTCTGGCCATCTCTGCCAGCTGATCTGCAAGCTGCATACGAGAACCCCAGAGATAAGATTCTGGTTTTTAAAGATGAAAACTTCTGGATGATCAGAGGATATGCTGTCTTGCCAGATTATCCCAAATCCATCCATACATTAGGTTTTCCAGGACGTGTGAAGAAAATAGATGCAGCCGTCTGTGATAAGACCACAAGAAAAACCTACTTCTTTGTGGGCATTTGGTGCTGGAGGTTTGATGAAATGACCCAAACCATGGACAAAGGATTCCCGCAGAGAGTGGTAAAACACTTTCCTGGAATCAGTATCCGTGTTGATGCTGCTTTCCAGTACAAAGGATTCTTCTTTTTCAGCCGTGGATCAAAGCAATTTGAATACAACATTAAGACAAAGAATATTACCCGAATCATGAGAACTAATACTTGGTTTCAATCCAAAGAACCAAAGAACTCCTCATTTGGTTTTGATATCAACAAGGAAAAAGCACATTCAGGAGGCATAAAGATATTGTATCATAAGAGTTTAAGCTTGTTTATTTTTGGTATTGTTCATTTGCTGAAAAACACTTCTATTTATCAATAAATTCATAGACCTAAAATAAACCTCAACAGGTCTTTTAATATAAATTCTGCTTCAAAATAGAATAAAACCATTCTTTAACAACAAGTTGCTGGTCCTAGTTCTAAATATCCAAATTCAATGGCCATTTTGAGCTGCCTGATTCTTTTAATAGGAAGTTATTATGTAGAAACAAAAATCTCTGACTGTACTTTAAGCCTATTTCATGCTTTGTGGACTTGGAGAAGACATGTCTTATAACTGAATACTGAAACATTTATTAAACCAATCTTTAGCATTCTAAThe following amino acid sequence <SEQ ID NO. 4> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 1:MKRLLLLCLFFITFSSAFPLVRMTENEENMOLAOAYLNOFYSLEIEGNHLVQSKNRSLIDDKIREMQAFFGLTVTGKLDSNTLEIMKTPRCGVPDVGQYGYTLPGWRKYNLTYRIINYTPDMARAAVDEAIQEGLEVWSKVTPLKFTKISKGIADIMIAFRTRVHGRCPRYFDGPLGVLGHAFPPGPGLGGDTHFDEDENWTKDGAGFNLFLVAAHEFGHALGLSHSNDQTALMFPNYVSLDPRKYPLSQDDINGIQSIYGGLPKVPAKPKEPTIPHACDPDLTFDAITTFRREVMFFKGRHLWRIYYDITDVEFELIASFWPSLPADLQAAYENPRDKILVFKDENFWMIRGYAVLPDYPKSIHTLGFPGRVKKIDAAVCDKTTRKTYFFVGIWCWRFDEMTQTMDKGFPQRVVKHFPGISIRVDAAFQYKGFFFFSRGSKQFEYNIKTKNITRIMRTNTWFQCKEPKNSSFGFDINKEKAHSGGIKILYHKSLSLFIFGIVHLLKNTSIYQ The following DNA sequence MMPU9 <SEQ ID NO. 2> wasidentified in H. sapiens:GACAAATGAGGGTTTGGCATGCAGCTCGTCATCTTAAGAGTTACTATCTTCTTGCCCTGGTGTTTCGCCGTTCCAGTGCCCCCTGCTGCAGACCATAAAGGATGGGACTTTGTTGAGGGCTATTTCCATCAATTTTTCCTGACCAAGAAGGAGTCGCCACTCCTTACCCAGGAGACACAAACACAGCTCCTGCAACAATTCCATCGGAATGGGACAGACCTACTTGACATGCAGATGCATGCTCTGCTACACCAGCCCCACTGTGGGGTGCCTGATGGGTCCGACACCTCCATCTCGCCAGGAAGATGCAAGTGGAATAAGCACACTCTAACTTACAGGATTATCAATTACCCACATGATATGAAGCCATCCGCAGTGAAAGACAGTATATATAATGCAGTTTCCATCTGGAGCAATGTGACCCCTTTGATATTCCAGCAAGTGCAGAATGGAGATGCACACATCAAGGTTTCTTTCTGGCAGTGGGCCCATGAAGATGGTTGGCCCTTTGATGGGCCAGGTGGTATCTTAGGCCATGCCTTTTTACCAAATTCTGGAAATCCTGGAGTTGTCCATTTTGACAAGAATGAACACTGGTCAGCTTCAGACACTGGATATAATCTGTTCCTGGTTGCAACTCATGAGATTGGGCATTCTTTGGGCCTGCAGCACTCTGGGAATCAGAGCTCCATAATGTACCCCACTTACTGGTATCACGACCCTAGAACCTTCCAGCTCAGTGCCGATGATATCCAAAGGATCCAGCATTTGTATGGAGAAAAATGTTCATCTGACATACCTTAATGTTAGCACAGAGGACTTATTCAACCTGTCCTTTCAGGGAGTTTATTGGAGGATCAAAGAACTGAAAGCACTAGAGCAGCCTTGGGGACTGCTAGGATGAAGCCCTAAAGAATGCAACCTAGTCAGGTTAGCTGAACCGACACTCAAAACGCTACTGAGTCACAATAAAGATTGTTTTAAAGAGT Thefollowing amino acid sequence <SEQ ID NO. 5> is the predicted amino acidsequence derived from the DNA sequence of SEQ ID NO. 2:MQLVILRVTIFLPWCFAVPVPPAADHKGWDFVEGYFHQFFLTKKESPLLTQETQTQLLQQFHRNGTDLLDMQMHALLHQPHCGVPDGSDTSISPGRCKWNKHTLTYRIINYPHDMKPSAVKDSIYNAVSIWSNVTPLTFQQVQNGDADIKVSFWQWAHEDGWPFDGPGGILGHAFLPNSGNPGVVHFDKNEHWSASDTGYNLFLVATHEIGHSLGLQHSGNQSSIMYPTYWYHDPRTFQLSADDIQRIQHLYGEKCSSD The following DNAsequence MMPU1O <SEQ ID NO. 3> was identified in H. sapiens:GCTCCCCGAGCCGGGCTGCACCGGAGGCGGCGAGATCGTCCCGCGCGTCGGCCTCCTGCTGCGCGCCCTGCAGCTGCTACTGTGGGGCCACCTGGACGCCCAGCCCGCGGAGCGCGGAGGCCAGGAGCTGCGCAAGGAGGCGGAGGCATTCCTAGAGAAGTACGGATACCTCAATGAACAGGTCCCCAAAGCTCCCACCTCCACTCGATTCAGCGATGCCATCAGAGCGTTTCAGTGGGTGTCCCAGCTACCTGTCAGCGGCGTGTTGGACCGCGCCACCCTGCGCCAGATGACTCGTCCCCGCTGCGGGGTTACAGATACCAACAGTTATGCGGCCTGGGCTGAGAGGATCAGTGACTTGTTTGCTAGACACCGGACCAAAATGAGGCGTAAGAAACGCTTTGCAAAGCAAGGTAACAAATGGTACAAGCAGCACCTCTCCTACCGCCTGGTCAACTGCCCTGAGCATCTCCGGAGCCGGCAGTTCGGGGCGCCGTGCGCGCCGCCTTCCAGTTGTGGAGCAACGTCTCAGCGCTGGAGTTCTGGGAGGCCCCAGCCACAGGCCCCGCTGACATCCGGCTCACCTTCTTCCAAGGGGACCACAACGATGGGCTGGGCAATGCCTTTGATGGCCCAGGGGGCGCCCTGCCGCACGCCTTTCCTGCCCCGCCGCGGCGAAGCGCACTTCGACCAAGATGAGCGCTGGTCCCTGAGCCGCCGCCGCGGGCGCAACCTGTTCGTCGTGCTGGCGCACGAGATCGCTCACACGCTTGGCCTCACCCACTCGCCCGCGCCGCGCGCGCTCATGCCGCCCTACTACAAGAGGCTGGGCCGCGACGCGCTGCTCAGCTGGGACGACGTGCTGGCCGTGCAGAGCCTGTATGGCAAGCCCCTAGGGGGCTCAGTCGCCGTCCAGCTCCCAGGAAAGCTGTTCACTGACTTTGAGACCTGGGACTCCTACAGCCCCCAAGGAAGGCGCCCTGAAACGCAGGGCCCTAAATACTGCCACTCTTCCTTCGATGCCATCACTGTAGACAGGCAACAGCAACTGTACATTTTTAAAGGGAGCCATTTCTGCGAGGTGGCAGCTGATGGCAACGTCTCAGAGCCCCGTCCACTGCAGGAAAGATGGGTCGGGCTGCCCCCCAACATTGAGGCTGCGGCAGTGTCATTGAATGATGGAGATTTCTACTTCTTCAAAGGGGGTCGATGCTGGAGGTTCCGGGGCCCCAAGCCAGTGTGGGGTCTCCCACAGCTGTGCCGGGCAGGGGGCCTGCCCCGCCATCCTGACGCCGCCCTCTTCTTCCCTCCTCTGCGCCGCCTCATCCTCTTCAAGCGTGCCCGCTACTACGTGCTGGCCCGAGCGGGACTGCAAGTGGAGCCCTACTACCCCCGAAGTCTGCAGGACTGGGGAGCCATCCCTGAGGAGCTCAGCGGCGCCCTGCCGAGGCCCGATGCCTCCATCATCTTCTTCCGAGATGACCGCTACTGGCGCCTCGACCAGGCCAAACTGCAGGCAACCACCTCGGGCCGCTGGGCCACCGACCTGCCCTGGATGGGCTGCTGGCATGCCAACTCGGGGAGCGCCCTGTTCTGA The following amino acid sequence<SEQ ID NO. 6> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 3:MVARVGLLLRALOLLLWGHLDAQPAERGGQELRKEAEAFLEKYGYLNEQVPKAPTSTRFSDAIRAFQWVSQLPVSGVLDRATLRQMTRPRCGVTDTNSYAAWAERISDLFARHRTKMRRKKRFAKQGNKWYKQHLSYRLVNWPEHLRSRQFGAPCAPPSSCGATSQRWSSGRPQPQAPLTSGSPSSKGTTTMGWAMPLMAQGAPWRTPFLPRRGEAHFDQDERWSLSRRRGRNLFVVLAHEIGHTLGLTHSPAPRALMAPYYKRLGRDALLSWDDVLAVQSLYGKPLGGSVAVOLPGKLFTDFETWDSYSPQGRRPETQGPKYCHSSFDAITVDRQQQLYIFKGSHFWEVAADGNVSEPRPLQERWVGLPPNIEAAAVSLNDGDFYFFKGGRCWRFRGPKPVWGLPQLCRAGGLPRHPDAALFFPPLRRLILFKGARYYVLARGGLQVEPYYPRSLQDWGGIPEEVSGALPRPDGSIIFFRDDRYWRLDQAKLQATTSGRWATELPWNGCWHANSGSALF The following DNA sequence <SEQ ID NO. 7> wasidentified in H. sapiens:ggcacgagcatgcagctcgtcatcttaagagttactatcttcttgccctggtgtttcgccgttccagtgccccctgctgcagaccataaaggatgggactttgttgagggctatttccatcaatttttcctgaccgagaaggagtcgccactccttacccaggagacacaaacacagctcctgcaacaattccatcggaatgggacagacctacttgacatgcagatgcatgctctgctacaccagccccactgtggggtgcctgatgggtccgacacctccatctcgccaggaagatgcaagtggaataagcacactctaacttacaggattatcaattacccacatgatatgaagccatccgcagtgaaagacagtatatataatgcagtttccatctggagcaatgtgacccctttgatattccagcaagtgcagaatggagatgcagacatcaaggtttctttctggcagtgggcccatgaagatggttggccctttgatgggceaggtggtatcttaggccatgcctttttaccaaattctggaaatcctggagttgtccattttgacaagaatgaacactggtcagcttcagacactggatataatctgttcctggttgcaactcatgagattgggcattctttgggcctgcagcactctgggaatcagagctccataatgtaccccacttactggtatcacgaccctagaaccttccagctcagtgccgatgatatccaaaggatccagcatttgtatggagaaaaatgttcatctgacataccttaatgttagcacagaggacttattcaacctgtctttcagggagtttattggaggatcaaagaactgaaagcactagagcagccttggggactgctaggatgaagccctaaagaatgcaacctagtcaggttagctgaaccgacactcaaaacgctactgagtcacaataaagattgttttaaagagtaaaaaaaaaaaaaaaaaaaa The following amino acid sequence <SEQ ID NO. 8> is the amino acidsequence derived from the DNA sequence of SEQ ID NO. 7:MVRVTWCAVVAADHKGWDVGYHTKSTTTHRNGTDDMMHAHHCGVDGSDTSSGRCKWNKHTTYRNYHDMKSAVKDSYNAVSWSNVTVNGDADKVSWWAHDGWDGGGGHANSGNGVVHDKNHWSASDTGYNVATHGHSGHSGNSSNYTYWYHDRTSADDRHYGKCSSD

EXAMPLE 2

Cloning of MMP cDNA

cDNAs may be sequenced directly using an AB1377 or AB1373Afluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division,PE/ABD, Foster City, Calif.) and the ABI PRISM Ready Dye-DeoxyTerminator kit with Taq FS polymerase. Each ABI cycle sequencingreaction contains about 0.5 μg of plasmid DNA. Cycle-sequencing isperformed using an initial denaturation at 98° C. for 1 min, followed by50 cycles: 98° C. for 30 sec, annealing at 50° C. for 30 sec, andextension at 60° C. for 4 min. Temperature cycles and times arecontrolled by a Perkin-Elmer 9600 thermocycler. Extension products arepurified using Centriflex gel filtration (Advanced Genetic TechnologiesCorp., Gaithersburg, Md.). Each reaction product is loaded by pipetteonto the column, which is then centrifuged in a swinging bucketcentrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4min at room temperature. Column-purified samples are dried under vacuumfor about 40 min and then dissolved in 5 μl of a DNA loading solution(83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran). Thesamples are then heated to 90° C. for three minutes and loaded into thegel sample wells for sequence analysis by the ABI377 sequencer. Sequenceanalysis is performed by importing ABI373A files into the Sequencherprogram (Gene Codes, Ann Arbor, Mich.). Generally, sequence reads of 700bp are obtained. Potential sequencing errors are minimized by obtainingsequence information from both DNA strands and by re-sequencingdifficult areas using primers at different locations until allsequencing ambiguities are removed.

To isolate a cDNA clone encoding full length MMP, a DNA fragmentcorresponding to a nucleotide sequence of SEQ ID NOS:1-3, or a portionthereof, can be used as a probe for hybridization screening of a phagecDNA library. The DNA fragment is amplified by the polymerase chainreaction (PCR) method. The PCR reaction mixture of 50 ml containspolymerase mixture (0.2 mM dNTPs, 1×PCR Buffer and 0.75 ml Expand HighFidelity Polymerase (Roche Biochemicals)), 1 μg of 3206491 plasmid, 50pmoles of forward primer and 50 pmoles of reverse primer. The primersare preferably 10 to 25 nucleotides in length and are determined byprocedures well known to those skilled in the art. Amplification isperformed in an Applied Biosystems PE2400 thermocycler, using thefollowing program: 95° C. for 15 seconds, 52° C. for 30 seconds and 72°C. for 90 seconds; repeated for 25 cycles. The amplified product isseparated from the plasmid by agarose gel electrophoresis, and purifiedby Qiaquick™ gel extraction kit (Qiagen).

A lambda phage library containing cDNAs cloned into lambda ZAPIIphage-vector is plated with E. coli XL-1 blue host, on 15 cm LB-agarplates at a density of 50,000 pfu per plate, and grown overnight at 37°C.; (plated as described by Sambrook et al., supra). Phage plaques aretransferred to nylon membranes (Amersham Hybond N.J.), denatured for 2minutes in denaturation solution (0.5 M NaOH, 1.5 M NaCl), renatured for5 minutes in renaturation solution (1 M Tris pH 7.5, 1.5 M NaCl), andwashed briefly in 2×SSC (20×SSC: 3 M NaCl, 0.3 M Na-citrate). Filtermembranes are dried and incubated at 80° C. for 120 minutes tocross-link the phage DNA to the membranes.

The membranes are hybridized with a DNA probe prepared as describedabove. A DNA fragment (25 ng) is labeled with α-³²P-dCTP (NEN) usingRediprime™ random priming (Amersham Pharmacia Biotech), according to themanufacturer's instructions. Labeled DNA is separated fromunincorporated nucleotides by S200 spin columns (Amersham PharmaciaBiotech), denatured at 95° C. for 5 minutes and kept on ice. TheDNA-containing membranes (above) are pre-hybridized in 50 ml ExpressHyb™(Clontech) solution at 68° C. for 90 minutes. Subsequently, the labeledDNA probe is added to the hybridization solution, and the probe is leftto hybridize to the membranes at 68° C. for 70 minutes. The membranesare washed five times in 2×SSC, 0.1% SDS at 42° C. for 5 minutes each,and finally washed for 30 minutes in 0.1×SSC, 0.2% SDS. Filters areexposed to Kodak XAR film (Eastman Kodak Company, Rochester, N.Y., USA)with an intensifying screen at −80° C. for 16 hours. One positive colonyis isolated from the plates, and replated with about 1000 pfu on a 15 cmLB plate. Plating, plaque lift to filters and hybridization areperformed as described above. About four positive phage plaques areisolated from this secondary screening.

cDNA containing plasmids (pBluescript SK-) are rescued from the isolatedphages by in vivo excision by culturing XL-1 blue cells co-infected withthe isolated phages and with the Excision helper phage, as described bymanufacturer (Stratagene). XL-blue cells containing the plasmids areplated on LB plates and grown at 37° C. for 16 hours. Colonies (18) fromeach plate are replated on LB plates and grown. One colony from eachplate is stricken onto a nylon filter in an ordered array, and thefilter is placed on a LB plate to raise the colonies. The filter is thenhybridized with a labeled probe as described above. About three positivecolonies are selected and grown up in LB medium. Plasmid DNA is isolatedfrom the three clones by Qiagen Midi Kit™ (Qiagen) according to themanufacturer's instructions. The size of the insert is determined bydigesting the plasmid with the restriction enzymes NotI and SalI, whichestablishes an insert size. The sequence of the entire insert isdetermined by automated sequencing on both strands of the plasmids.

EXAMPLE 3

Subcloning of the Coding Region of MMP via PCR

Additional experiments may be conducted to subclone the coding region ofMMP and place the isolated coding region into a useful vector. Twoadditional PCR primers are designed based on the coding region of MMP,corresponding to either end. To protect against exonucleolytic attackduring subsequent exposure to enzymes, e.g., Taq polymerase, primers areroutinely synthesized with a protective run of nucleotides at the 5′ endthat are not necessarily complementary to the desired target.

PCR is performed in a 50 μl reaction containing 34 μl H₂O, 5 μl 10×TTbuffer (140 mM ammonium sulfate, 0.1% gelatin, 0.6 M Tris-tricine, pH8.4), 5 μl 15 mM MgSO₄, 2 μl dNTP mixture (dGTP, dATP, dTTP, and dCTP,each at 10 mM), 3 μl genomic phage DNA (0.25 μg/μl), 0.3 μl Primer 1 (1μg/μl), 0.3 μl Primer 2 (1 μg/μl), 0.4 μl High Fidelity Taq polymerase(Boehringer Mannheim). The PCR reaction is started with 1 cycle of 94°C. for 2 minutes; followed by 25 cycles at 94° C. for 30 seconds, 55° C.for 30 seconds, and 72° C. for 1.3 minutes.

The contents from the PCR reaction are loaded onto a 2% agarose gel andfractionated. The DNA band of expected size is excised from the gel,placed in a GenElute Agarose spin column (Supelco), and spun for 10minutes at maximum speed in a microfuge tube placed in amicrocentrifuge. The eluted DNA is precipitated with ethanol andresuspended in 6 μl H₂O for ligation.

The PCR-amplified DNA fragment containing the coding region is clonedinto pCR2.1 using a protocol standard in the art. In particular, theligation reaction consists of 6 μl of MMP DNA, 1 μl 10×ligation buffer,2 μl pCR2.1 (25 ng/μl, Invitrogen), and 1 μl T4 DNA ligase (Invitrogen).The reaction mixture is incubated overnight at 14° C., and the reactionis then terminated by heating at 65° C. for 10 minutes. Two microlitersof the ligation reaction are transformed into One Shot cells(Invitrogen) and plated onto ampicillin plates. A single colonycontaining a recombinant pCR2.1 bearing an insert is used to inoculate a5 ml culture of LB medium. Plasmid DNA is purified using the ConcertRapid Plasmid Miniprep System (GibcoBRL) and sequenced. Followingconfirmation of the sequence, a 50 ml culture of LB medium is inoculatedwith the transformed One Shot cells, cultured, and processed using aQiagen Plasmid Midi Kit to yield purified pCR-MMP.

EXAMPLE 4

Hybridization Analysis to Demonstrate MMP Expression in Various Tissues

The expression of MMP in mammals, such as the rat or mouse, isinvestigated by in situ hybridization histochemistry as described inBertilsson et al. (supra). Tissue sections are thaw-mounted ontosilanized, nuclease-free slides (CEL Associates, Inc., Houston, Tex.),and stored at −80° C. Sections are processed starting with post-fixationin cold 4% paraformaldehyde, rinsed in cold phosphate-buffered saline(PBS), acetylated using acetic anhydride in triethanolamine buffer, anddehydrated through a series of alcohol washes in 70%, 95%, and 100%alcohol at room temperature. Subsequently, sections are delipidated inchloroform, followed by rehydration through successive exposure to 100%and 95% alcohol at room temperature. Microscope slides containingprocessed cryosections are allowed to air dry prior to hybridization.

An MMP-specific probe is generated using PCR. Following PCRamplification, the fragment is digested with restriction enzymes andcloned into pBluescript II cleaved with the same enzymes. For productionof a probe specific for the sense strand of MMP, the MMP clone inpBluescript II is linearized with a suitable restriction enzyme, whichprovides a substrate for labeled run-off transcripts (i.e., cRNAriboprobes) using the vector-borne T7 promoter and commerciallyavailable T7 RNA polymerase. A probe specific for the antisense strandof MMP is also readily prepared using the MMP clone in pBluescript II bycleaving the recombinant plasmid with a suitable restriction enzyme togenerate a linearized substrate for the production of labeled run-offcRNA transcripts using the T3 promoter and cognate polymerase. Theriboprobes are labeled with [³⁵S]-UTP to yield a specific activity ofabout 0.40×10⁶ cpm/pmol for antisense riboprobes and about 0.65×10⁶cpm/pmol for sense-strand riboprobes Each riboprobe is subsequentlydenatured and added (2 pmol/ml) to hybridization buffer which contained50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA,1×Denhardt's Solution, and 10 mM dithiothreitol. Microscope slidescontaining sequential brain cryosections are independently exposed to 45μl of hybridization solution per slide and silanized cover slips areplaced over the sections being exposed to hybridization solution.Sections are incubated overnight (15-18 hours) at 52° C. to allowhybridization to occur. Equivalent series of cryosections are exposed tosense or antisense MMP-specific cRNA riboprobes.

Following the hybridization period, coverslips are washed off the slidesin 1×SSC, followed by RNase A treatment involving the exposure of slidesto 20 μg/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5M EDTA, and 0.5 M NaCl for 45 minutes at 37° C. The cryosections arethen subjected to three high-stringency washes in 0.1×SSC at 52° C. for20 minutes each. Following the series of washes, cryosections aredehydrated by consecutive exposure to 70%, 95%, and 100% ammoniumacetate in alcohol, followed by air drying and exposure to Kodak BioMaxMR-1 film. After 13 days of exposure, the film is developed. Based onthese results, slides containing tissue that hybridized, as shown byfilm autoradiograms, are coated with Kodak NTB-2 nuclear track emulsionand the slides are stored in the dark for 32 days. The slides are thendeveloped and counterstained with hematoxylin. Emulsion-coated sectionsare analyzed microscopically to determine the specificity of labeling.The signal is determined to be specific if autoradiographic grains(generated by antisense probe hybridization) are clearly associated withcresyl violate-stained cell bodies. Autoradiographic grains foundbetween cell bodies indicates non-specific binding of the probe.

A PCR-based system (RapidScan™ Gene Expression Panel, OriGeneTechnologies, Rockville, Md.) may be used to generate a comprehensiveexpression profile of the putative MMP in human tissue, and in humanbrain regions. The RapidScan Expression Panel is comprised offirst-strand cDNAs from various human tissues and brain regions thatwere serially diluted over a 4-log range and arrayed into a multi-wellPCR plate. Human tissues arrayed may include: brain, heart, kidney,spleen, liver, colon, lung, small intestine, muscle, stomach, testis,placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary,uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.Human brain regions arrayed may include: frontal lobe, temporal lobe,cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala,thalamus, hypothalamus, pons, medulla and spinal cord.

Expression of the NMP in the various tissues is detected by using PCRprimers designed based on the available sequence of the protein thatwill prime the synthesis of a fragment of predetermined size in thepresence of the appropriate cDNA. The dilution range of cDNA depositedon the plate (e.g., 4-log) is chosen to ensure that the amplificationreaction is within the linear range and, hence, will facilitatesemi-quantitative determination of relative mRNA accumulation in thevarious tissues or brain regions examined.

Expression of MMP in different tissues provides an indication thatmodulators of MMP activity have utility for treating various disorders,including but not limited to metabolic diseases and disorders (e.g.,type 2 diabetes, obesity, cardiovascular, dyslipidemias, adipogenesis,retinopathies, neuropathies, nephropathies etc.), proliferative diseasesand cancers (e.g., different cancers such as breast, colon, lung, etc.,tumor growth, tumor invasion, and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc.), hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.), CNS disorders (e.g., degenerative disorders such as Parkinson's,Alzheimer's, etc.), inflammatory conditions (e.g., Chron's disease,arthritis), diseases related to cell differentiation and homeostasis,cardiomyopathy, atherosclerosis, thromboembolic diseases, Sjögren'ssyndrome, renal failure, periodontal diseases, retinalneovascularization, wound healing, and neurodegenerative diseasesincluding, for example, Alzheimer's disease, multiple sclerosis,Parkinson's disease, and motoneuron disease, among others. Use of MMPmodulators, including MMP ligands (activators and repressors) andanti-MMP antibodies, to treat individuals having such disease states isintended as an aspect of the invention.

EXAMPLE 5

Northern Blot Analysis

Northern blots are performed to examine the expression of mRNA. Thesense orientation oligonucleotide and the antisense-orientationoligonucleotide, described above, are used as primers to amplify aportion of the MMP cDNA sequence of a nucleotide sequence of SEQ ID NOS:1-3.

Multiple human tissue northern blot from Clontech are hybridized withthe probe according to the recommendations of the manufacturer, and asdescribed by Bertilsson (supra). The probe is labeled with α-³²P-dCTP byRediprime™ DNA labeling system (Amersham Pharmacia), purified on NickColumn™ (Amersham Pharmacia) and added to the hybridization solution.The filters are washed several times at 42° C. in 0.2×SSC, 0.1% SDS.Filters are exposed to Kodak XAR film (Eastman Kodak Company, Rochester,N.Y., USA) with intensifying screen at −80° C.

EXAMPLE 6

Recombinant Expression of MMP in Eukaryotic Host Cells

A. Expression of MMP in Mammalian Cells

To produce MMP protein, a MMP-encoding polynucleotide is expressed in asuitable host cell using a suitable expression vector and standardgenetic engineering techniques. For example, the MMP-encoding sequencedescribed in Example 1 is subcloned into the commercial expressionvector pzeoSV2 (Invitrogen, San Diego, Calif.) and transfected intoChinese Hamster Ovary (CHO) cells using the transfection reagent FuGENE6 or Dosper (Boehringer-Mannheim) and the transfection protocol providedin the product insert. Other eukaryotic cell lines, including humanembryonic kidney (HEK 293), human colon cancer cells (CaCo-2), and COScells, are suitable as well. Cells stably expressing MMP are selected bygrowth in the presence of 100 μg/ml zeocin (Stratagene, LaJolla,Calif.). Optionally, MMP may be purified from the cells using standardchromatographic techniques. To facilitate purification, antisera israised against one or more synthetic peptide sequences that correspondto portions of the MMP amino acid sequence, and the antisera is used toaffinity purify MMP. The MMP also may be expressed in-frame with a tagsequence (e.g., polyhistidine, hemagluttinin, FLAG) to facilitatepurification. Moreover, it will be appreciated that many of the uses forMMP polypeptides, such as assays described below, do not requirepurification of MMP from the host cell.

B. Expression of MMP in HEK-293 Cells

For expression of MMP in mammalian cells 293 (transformed human, primaryembryonic kidney cells), a plasmid bearing the relevant MMP codingsequence is prepared, using vector pSecTag2A (Invitrogen). VectorpSecTag2A contains the murine IgK chain leader sequence for secretion,the c-myc epitope for detection of the recombinant protein with theanti-myc antibody, a C-terminal polyhistidine for purification withnickel chelate chromatography, and a Zeocin resistant gene for selectionof stable transfectants. The forward primer for amplification of thisMMP cDNA is determined by routine procedures and preferably contains a5′ extension of nucleotides to introduce the HindIII cloning site andnucleotides matching the MMP sequence. The reverse primer is alsodetermined by routine procedures and preferably contains a 5′ extensionof nucleotides to introduce a XhoI restriction site for cloning andnucleotides corresponding to the reverse complement of the MMP sequence.The PCR conditions use 55° C. as the annealing temperature. The PCRproduct is gel purified and cloned into the HindIII-XhoI sites of thevector.

The DNA is purified using Qiagen chromatography columns and transfectedinto 293 cells using DOTAP transfection media (Boehringer Mannheim,Indianapolis, Ind.). Transiently transfected cells are tested forexpression after 24 hours of transfection, using western blots probedwith anti-His and anti-MMP peptide antibodies. Permanently transfectedcells are selected with Zeocin and propagated. Production of therecombinant protein is detected from both cells and media by westernblots probed with anti-His, anti-Myc or anti-MMP peptide antibodies.

C. Expression of MMP in COS Cells

For expression of the MMP in COS7 cells, a polynucleotide moleculehaving a nucleotide sequence of SEQ ID NOS: 1-3 can be cloned intovector p3-CI. This vector is a pUC18-derived plasmid that contains theHCMV (human cytomegalovirus) promoter-intron located upstream from thebGH (bovine growth hormone) polyadenylation sequence and a multiplecloning site. In addition, the plasmid contains the dhrf (dihydrofolatereductase) gene which provides selection in the presence of the drugmethotrexane (MTX) for selection of stable transformants.

The forward primer is determined by routine procedures and preferablycontains a 5′ extension which introduces an XbaI restriction site forcloning, followed by nucleotides which correspond to a nucleotidesequence of SEQ ID NOS: 1-3. The reverse primer is also determined byroutine procedures and preferably contains 5′-extension of nucleotideswhich introduces a SalI cloning site followed by nucleotides whichcorrespond to the reverse complement of a nucleotide sequence of SEQ IDNOS: 1-3.

The PCR consists of an initial denaturation step of 5 min at 95° C., 30cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58° C. and30 sec extension at 72° C., followed by 5 min extension at 72° C. ThePCR product is gel purified and ligated into the XbaI and SalI sites ofvector p3-CI. This construct is transformed into E. coli cells foramplification and DNA purification. The DNA is purified with Qiagenchromatography columns and transfected into COS 7 cells usingLipofectamine reagent from BRL, following the manufacturer's protocols.Forty-eight and 72 hours after transfection, the media and the cells aretested for recombinant protein expression.

MMP expressed from a COS cell culture can be purified by concentratingthe cell-growth media to about 10 mg of protein/ml, and purifying theprotein by, for example, chromatography. Purified MMP is concentrated to0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane andstored at −80° C.

D. Expression of MMP in Insect Cells

For expression of MMP in a baculovirus system, a polynucleotide moleculehaving a nucleotide sequence of SEQ ID NOS: 1-3 can be amplified by PCR.The forward primer is determined by routine procedures and preferablycontains a 5′ extension which adds the NdeI cloning site, followed byfollowed by nucleotides which correspond to a nucleotide sequence of SEQID NOS: 1-3. The reverse primer is also determined by routine proceduresand preferably contains a 5′ extension which introduces the KpnI cloningsite, followed by followed by nucleotides which correspond to thereverse complement of a nucleotide sequence of SEQ ID NOS: 1-3.

The PCR product is gel purified, digested with NdeI and KpnI, and clonedinto the corresponding sites of vector pACHTL-A (Pharmingen, San Diego,Calif.). The pAcHTL expression vector contains the strong polyhedrinpromoter of the Autographa californica nuclear polyhedrosis virus(AcMNPV), and a 6×His tag upstream from the multiple cloning site. Aprotein kinase site for phosphorylation and a thrombin site for excisionof the recombinant protein precede the multiple cloning site is alsopresent. Of course, many other baculovirus vectors could be used inplace of pAcHTL-A, such as pAc373, pVL941 and pAcIM1. Other suitablevectors for the expression of MMP polypeptides can be used, providedthat the vector construct includes appropriately located signals fortranscription, translation, and trafficking, such as an in-frame AUG anda signal peptide, as required. Such vectors are described in Luckow etal., Virology 170:31-39, among others.

The virus is grown and isolated using standard baculovirus expressionmethods, such as those described in Summers et al. (A Manual of Methodsfor Baculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987)).

In a preferred embodiment, pAcHLT-A containing MMP gene is introducedinto baculovirus using the “BaculoGold” transfection kit (Pharmingen,San Diego, Calif.) using methods established by the manufacturer.Individual virus isolates are analyzed for protein production byradiolabeling infected cells with ³⁵S-methionine at 24 hours postinfection. Infected cells are harvested at 48 hours post infection, andthe labeled proteins are visualized by SDS-PAGE. Viruses exhibiting highexpression levels can be isolated and used for scaled up expression.

For expression of a MMP polypeptide in Sf9 cells, a polynucleotidemolecule having a nucleotide sequence of SEQ ID NOS: 1-3, can beamplified by PCR using the primers and methods described above forbaculovirus expression. The MMP cDNA is cloned into vector pAcHLT-A(Pharmingen) for expression in Sf9 insect. The insert is cloned into theNdeI and KpnI sites, after elimination of an internal NdeI site (usingthe same primers described above for expression in baculovirus). DNA ispurified with Qiagen chromatography columns and expressed in Sf9 cells.Preliminary Western blot experiments from non purified plaques aretested for the presence of the recombinant protein of the expected sizewhich reacted with the MMP-specific antibody. These results areconfirmed after further purification and expression optimization in HiG5cells.

EXAMPLE 7

Zymography

MMP protease activity is analyzed by substrate gel electrophoresis(zymography) in polyacrylamide gels containing 2 mg/ml gelatin or 1.5mg/ml casein. Samples are dissolved in modified Laemmli sample buffer[containing 2.5% (v/v) SDS without 2-mercaptoethanol] andelectrophoresed, without prior boiling, at 4° C. After removal of theSDS by washing in 2.5% (v/v) Triton X-100 in 50 mM Tris/HCl, pH 7.5, for1 h, the gels are incubated overnight at 37° C. in a buffer containing40 mM Tris/HCl, pH 7.5, and 10 mM CaCl₂. Staining with 0.5% (w/v)Coomassie Brilliant Blue (Bio-Rad, Richmond, Calif., U.S.A.) in 30%(v/v) isopropanol/10% (v/v) acetic acid followed by destaining with 30%isopropanol/10% acetic acid allows identification of gelatinolytic orcaseinolytic activity as clear zones in a blue background.

EXAMPLE 8

Antibodies to MMP

Standard techniques are employed to generate polyclonal or monoclonalantibodies to the MMP, and to generate useful antigen-binding fragmentsthereof or variants thereof, including “humanized” variants. Suchprotocols can be found, for example, in Sambrook et al. (1989) andHarlow et al. (Eds.), Antibodies A Laboratory Manual; Cold Spring HarborLaboratory; Cold Spring Harbor, N.Y. (1988). In one embodiment,recombinant MMP polypeptides (or cells or cell membranes containing suchpolypeptides) are used as antigen to generate the antibodies. In anotherembodiment, one or more peptides having amino acid sequencescorresponding to an immunogenic portion of MMP (e.g., 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) are used asantigen. The antigen may be mixed with an adjuvant or linked to a haptento increase antibody production.

A. Polyclonal or Monoclonal Antibodies

As one exemplary protocol, recombinant MMP or a synthetic fragmentthereof is used to immunize a mouse for generation of monoclonalantibodies (or larger mammal, such as a rabbit, for polyclonalantibodies). To increase antigenicity, peptides are conjugated toKeyhole Lympet Hemocyanin (Pierce), according to the manufacturer'srecommendations. For an initial injection, the antigen is emulsifiedwith Freund's Complete Adjuvant and injected subcutaneously. Atintervals of two to three weeks, additional aliquots of MMP antigen areemulsified with Freund's Incomplete Adjuvant and injectedsubcutaneously. Prior to the final booster injection, a serum sample istaken from the immunized mice and assayed by western blot to confirm thepresence of antibodies that immunoreact with MMP. Serum from theimmunized animals may be used as a polyclonal antisera or used toisolate polyclonal antibodies that recognize MMP. Alternatively, themice are sacrificed and their spleen removed for generation ofmonoclonal antibodies.

To generate monoclonal antibodies, the spleens are placed in 10 mlserum-free RPMI 1640, and single cell suspensions are formed by grindingthe spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine,1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/mlstreptomycin (RPMI) (Gibco, Canada). The cell suspensions are filteredand washed by centrifugation and resuspended in serum-free RPMI.Thymocytes taken from three naive Balb/c mice are prepared in a similarmanner and used as a Feeder Layer. NS-1 myeloma cells, kept in log phasein RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc.,Logan, Utah) for three days prior to fusion, are centrifuged and washedas well.

To produce hybridoma fusions, spleen cells from the immunized mice arecombined with NS-1 cells and centrifuged, and the supernatant isaspirated. The cell pellet is dislodged by tapping the tube, and 2 ml of37° C. PEG 1500 (50% in 75 mM BEPES, pH 8.0) (Boehringer-Mannheim) isstirred into the pellet, followed by the addition of serum-free RPMI.Thereafter, the cells are centrifuged, resuspended in RPMI containing15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine(HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×10⁶thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissueculture plates (Corning, Corning N.Y.).

On days 2, 4, and 6 after the fusion, 100 μl of medium is removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,the fusions are screened by ELISA, testing for the presence of mouse IgGthat binds to MMP. Selected fusion wells are further cloned by dilutionuntil monoclonal cultures producing anti-MMP antibodies are obtained.

B. Humanization of Anti-MMP Monoclonal Antibodies

The expression pattern of MMP as reported herein suggests therapeuticindications for MMP inhibitors (repressors). MMP-neutralizing antibodiescomprise one class of therapeutics useful as MMP repressors. Followingare protocols to improve the utility of anti-MMP monoclonal antibodiesas therapeutics in humans by “humanizing” the monoclonal antibodies toimprove their serum half-life and render them less immunogenic in humanhosts (i.e., to prevent human antibody response to non-human anti-MMPantibodies).

The principles of humanization have been described in the literature andare facilitated by the modular arrangement of antibody proteins. Tominimize the possibility of binding complement, a humanized antibody ofthe IgG4 isotype is preferred.

For example, a level of humanization is achieved by generating chimericantibodies comprising the variable domains of non-human antibodyproteins of interest with the constant domains of human antibodymolecules. (See, e.g., Morrison et al., Adv. Immunol., 44:65-92 (1989)).The variable domains of MMP-neutralizing anti-MMP antibodies are clonedfrom the genomic DNA of a B-cell hybridoma or from cDNA generated frommRNA isolated from the hybridoma of interest. The V region genefragments are linked to exons encoding human antibody constant domains,and the resultant construct is expressed in suitable mammalian hostcells (e.g., myeloma or CHO cells).

To achieve an even greater level of humanization, only those portions ofthe variable region gene fragments that encode antigen-bindingcomplementarity determining regions (CDR) of the non-human monoclonalantibody genes are cloned into human antibody sequences. (See, e.g.,Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-36 (1988); andTempest et al., Bio/Technology 9:266-71 (1991)). If necessary, theβ-sheet framework of the human antibody surrounding the CDR3 regionsalso is modified to more closely mirror the three dimensional structureof the antigen-binding domain of the original monoclonal antibody. (SeeKettleborough et al., Protein Engin., 4:773-783 (1991); and Foote etal., J. Mol. Biol., 224:487-499 (1992)).

In an alternative approach, the surface of a non-human monoclonalantibody of interest is humanized by altering selected surface residuesof the non-human antibody, e.g., by site-directed mutagenesis, whileretaining all of the interior and contacting residues of the non-humanantibody. See Padlan, Molecular Immunol., 28(4/5):489-98 (1991).

The foregoing approaches are employed using MMP-neutralizing anti-MMPmonoclonal antibodies and the hybridomas that produce them to generatehumanized MMP-neutralizing antibodies that are useful as therapeutics totreat or palliate conditions wherein MMP expression or ligand-mediatedMMP activity is detrimental.

C. Human MMP-Neutralizing Antibodies from Phage Display

Human MMP-neutralizing antibodies are generated by phage displaytechniques such as those described in Aujame et al., Human Antibodies8(4): 155-168 (1997); Hoogenboom, TIBTECH 15:62-70 (1997); and Rader etal., Curr. Opin. Biotechnol. 8:503-508 (1997), all of which areincorporated by reference. For example, antibody variable regions in theform of Fab fragments or linked single chain Fv fragments are fused tothe amino terminus of filamentous phage minor coat protein pIII.Expression of the fusion protein and incorporation thereof into themature phage coat results in phage particles that present an antibody ontheir surface and contain the genetic material encoding the antibody. Aphage library comprising such constructs is expressed in bacteria, andthe library is screened for MMP-specific phage-antibodies using labeledor immobilized MMP as antigen-probe.

D. Human MMP-neutralizing Antibodies from Transgenic Mice

Human NMP-neutralizing antibodies are generated in transgenic miceessentially as described in Bruggemann et al., Immunol. Today17(8):391-97 (1996) and Bruggemann et al., Curr. Opin. Biotechnol.8:455-58 (1997). Transgenic mice carrying human V-gene segments ingermline configuration and that express these transgenes in theirlymphoid tissue are immunized with a MMP composition using conventionalimmunization protocols. Hybridomas are generated using B cells from theimmunized mice using conventional protocols and screened to identifyhybridomas secreting anti-MMP human antibodies (e.g., as describedabove).

EXAMPLE 9

Assays to Assess MMP Activity and to Identify Modulators of MMP Activity

A. Synthetic Fluorogenic Peptide Substrate Cleavage Assays

Fluorogenic peptide substrates may be used to test proteinase activityas well as inhibitors of proteinase activity. Such peptide substratesmay be purchased, for example, from BACHEM Bioscience Inc, who offerseveral MMP substrates and various enzyme substrates, for example asubstrate of tumor necrosis factor-α (TNF-α) converting enzyme (TACE).Substrates are prepared as 50-500 μM stock solutions in 1:1 dimethylsulfoxide (DMSO) and water. Fluorescent assays are performed atλ_(excitation)=328 nm and λ_(emmission)=393 nm using a luminescencespectrometer equipped with a constant-temperature water bath. Therelationship between fluorescence units and nanomoles of productproduced is determined from the fluorescence value obtained when all thesubstrate is hydrolyzed.

B. Kinetic Parameters

Assays for obtaining kinetic parameters are performed at 25° C. in 10 mMCaCl₂ 0.2 M NaCl and 0.05% Brij-35 in 50 mM BEPES, pH 7.5 over thesubstrate concentration range 1-4 μM range and enzyme concentrationrange 0.06-50 nM under steady-state conditions. Stock solutions of MMPsare diluted to 1-500 nM by adding 50 mM HEPES buffer containing 10 mMCaCl₂ 0.2 M NaCl, and 0.05% Brij-35 or 50 mM Tricine buffer with thesame constituents. A typical assay is carried out by incubating 186 μLof buffer solution and 4 μL of substrate solution in an assay cuvettefor at least for 15 min. at 25° C., and then adding 10 μL of enzymesolution into the assay cuvette. Initial hydrolysis rates are monitoredfor 10-30 min.

C. Inhibition Studies

Modulators of MMP activity may be studied using the above assay. Theactivity of MMPs in the absence of potential modulating compounds iscompared to the activity of MMPs in the presence of varyingconcentrations of potential modulating compounds.

Among the modulators that can be identified by these assays are naturalligand compounds of the MMP; synthetic analogs and derivatives ofnatural ligands; antibodies, antibody fragments, and/or antibody-likecompounds derived from natural antibodies or from antibody-likecombinatorial libraries; and/or synthetic compounds identified byhigh-throughput screening of libraries; and the like. All modulatorsthat bind MMP are useful for identifying MMPs in tissue samples (e.g.,for diagnostic purposes, pathological purposes, and the like). Activatorand repressor modulators are useful for up-regulating anddown-regulating MMP activity, respectively, to treat disease statescharacterized by abnormal levels of MMP activity. The assays may beperformed using single putative modulators, and/or may be performedusing a known activator in combination with candidate repressors (orvisa versa).

Some of the preferred embodiments of the invention described above areoutlined below and include, but are not limited to, the followingembodiments. As those skilled in the art will appreciate, numerouschanges and modifications may be made to the preferred embodiments ofthe invention without departing from the spirit of the invention. It isintended that all such variations fall within the scope of theinvention.

The entire disclosure of each publication cited herein is herebyincorporated by reference.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 8 <210> SEQ ID NO 1 <211> LENGTH: 1845<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1gcttcagctg aagaaagaga ggaatgaagc gccttctgct tctgtgtttg tt#ctttataa     60cattttcttc tgcatttccc ttagtccgga tgacggaaaa tgaagaaaat at#gcaactgg    120ctcaggcata tctcaaccag ttctactctc ttgaaataga agggaatcat ct#tgttcaaa    180gcaagaatag gagtctcata gatgacaaaa ttcgggaaat gcaagcattt tt#tggattga    240cagtgactgg aaaactggac tcaaacaccc ttgagatcat gaagacaccc ag#gtgtgggg    300tgcctgatgt gggccagtat ggctacaccc tccctgggtg gagaaaatac aa#cctcacct    360acagaataat aaactatact ccggatatgg cacgagctgc tgtggatgag gc#tatccaag    420aaggtttaga agtgtggagc aaagtcactc cactaaaatt caccaagatt tc#aaagggga    480ttgcagacat catgattgcc tttaggactc gagtccatgg tcggtgtcct cg#ctattttg    540atggtccctt gggagtgctt ggccatgcct ttcctcctgg tccgggtctg gg#tggtgaca    600ctcattttga tgaggatgaa aactggacca aggatggagc aggattcaac tt#gtttcttg    660tggctgctca tgaatttggt catgcactgg ggctctctca ctccaatgat ca#aacagcct    720tgatgttccc aaattatgtc tccctggatc ccagaaaata cccactttct ca#ggatgata    780tcaatggaat ccagtccatc tatggaggtc tgcctaaggt acctgctaag cc#aaaggaac    840ccactatacc ccatgcctgt gaccctgact tgacttttga cgctatcaca ac#tttccgca    900gagaagtaat gttctttaaa ggcaggcacc tatggaggat ctattatgat at#cacggatg    960ttgagtttga attaattgct tcattctggc catctctgcc agctgatctg ca#agctgcat   1020acgagaaccc cagagataag attctggttt ttaaagatga aaacttctgg at#gatcagag   1080gatatgctgt cttgccagat tatcccaaat ccatccatac attaggtttt cc#aggacgtg   1140tgaagaaaat agatgcagcc gtctgtgata agaccacaag aaaaacctac tt#ctttgtgg   1200gcatttggtg ctggaggttt gatgaaatga cccaaaccat ggacaaagga tt#cccgcaga   1260gagtggtaaa acactttcct ggaatcagta tccgtgttga tgctgctttc ca#gtacaaag   1320gattcttctt tttcagccgt ggatcaaagc aatttgaata caacattaag ac#aaagaata   1380ttacccgaat catgagaact aatacttggt ttcaatgcaa agaaccaaag aa#ctcctcat   1440ttggttttga tatcaacaag gaaaaagcac attcaggagg cataaagata tt#gtatcata   1500agagtttaag cttgtttatt tttggtattg ttcatttgct gaaaaacact tc#tatttatc   1560aataaattca tagacctaaa ataaacctca acaggtcttt taatataaat tc#tgcttcaa   1620aatagaataa aaccattctt taacaacaag ttgctggtcc tagttctaaa ta#tccaaatt   1680caatggccat tttgagctgc ctgattcttt taataggaag ttattatgta ga#aacaaaaa   1740tctctgactg tactttaagc ctatttcatg ctttgtggac ttggagaaga ca#tgtcttat   1800 aactgaatac tgaaacattt attaaaccaa tctttagcat tctaa   #                1845 <210> SEQ ID NO 2 <211> LENGTH: 989<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2gacaaatgag ggtttggcat gcagctcgtc atcttaagag ttactatctt ct#tgccctgg     60tgtttcgccg ttccagtgcc ccctgctgca gaccataaag gatgggactt tg#ttgagggc    120tatttccatc aatttttcct gaccaagaag gagtcgccac tccttaccca gg#agacacaa    180acacagctcc tgcaacaatt ccatcggaat gggacagacc tacttgacat gc#agatgcat    240gctctgctac accagcccca ctgtggggtg cctgatgggt ccgacacctc ca#tctcgcca    300ggaagatgca agtggaataa gcacactcta acttacagga ttatcaatta cc#cacatgat    360atgaagccat ccgcagtgaa agacagtata tataatgcag tttccatctg ga#gcaatgtg    420acccctttga tattccagca agtgcagaat ggagatgcag acatcaaggt tt#ctttctgg    480cagtgggccc atgaagatgg ttggcccttt gatgggccag gtggtatctt ag#gccatgcc    540tttttaccaa attctggaaa tcctggagtt gtccattttg acaagaatga ac#actggtca    600gcttcagaca ctggatataa tctgttcctg gttgcaactc atgagattgg gc#attctttg    660ggcctgcagc actctgggaa tcagagctcc ataatgtacc ccacttactg gt#atcacgac    720cctagaacct tccagctcag tgccgatgat atccaaagga tccagcattt gt#atggagaa    780aaatgttcat ctgacatacc ttaatgttag cacagaggac ttattcaacc tg#tcctttca    840gggagtttat tggaggatca aagaactgaa agcactagag cagccttggg ga#ctgctagg    900atgaagccct aaagaatgca acctagtcag gttagctgaa ccgacactca aa#acgctact    960 gagtcacaat aaagattgtt ttaaagagt         #                   #           989 <210> SEQ ID NO 3 <211> LENGTH: 1597<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3gctccccgag ccgggctgca ccggaggcgg cgagatggtc gcgcgcgtcg gc#ctcctgct     60gcgcgccctg cagctgctac tgtggggcca cctggacgcc cagcccgcgg ag#cgcggagg    120ccaggagctg cgcaaggagg cggaggcatt cctagagaag tacggatacc tc#aatgaaca    180ggtccccaaa gctcccacct ccactcgatt cagcgatgcc atcagagcgt tt#cagtgggt    240gtcccagcta cctgtcagcg gcgtgttgga ccgcgccacc ctgcgccaga tg#actcgtcc    300ccgctgcggg gttacagata ccaacagtta tgcggcctgg gctgagagga tc#agtgactt    360gtttgctaga caccggacca aaatgaggcg taagaaacgc tttgcaaagc aa#ggtaacaa    420atggtacaag cagcacctct cctaccgcct ggtgaactgg cctgagcatc tc#cggagccg    480gcagttcggg gcgccgtgcg cgccgccttc cagttgtgga gcaacgtctc ag#cgctggag    540ttctgggagg ccccagccac aggccccgct gacatccggc tcaccttctt cc#aaggggac    600cacaacgatg ggctgggcaa tgcctttgat ggcccagggg gcgccctggc gc#acgccttt    660cctgccccgc cgcggcgaag cgcacttcga ccaagatgag cgctggtccc tg#agccgccg    720ccgcgggcgc aacctgttcg tggtgctggc gcacgagatc ggtcacacgc tt#ggcctcac    780ccactcgccc gcgccgcgcg cgctcatggc gccctactac aagaggctgg gc#cgcgacgc    840gctgctcagc tgggacgacg tgctggccgt gcagagcctg tatgggaagc cc#ctaggggg    900ctcagtggcc gtccagctcc caggaaagct gttcactgac tttgagacct gg#gactccta    960cagcccccaa ggaaggcgcc ctgaaacgca gggccctaaa tactgccact ct#tccttcga   1020tgccatcact gtagacaggc aacagcaact gtacattttt aaagggagcc at#ttctggga   1080ggtggcagct gatggcaacg tctcagagcc ccgtccactg caggaaagat gg#gtcgggct   1140gccccccaac attgaggctg cggcagtgtc attgaatgat ggagatttct ac#ttcttcaa   1200agggggtcga tgctggaggt tccggggccc caagccagtg tggggtctcc ca#cagctgtg   1260ccgggcaggg ggcctgcccc gccatcctga cgccgccctc ttcttccctc ct#ctgcgccg   1320cctcatcctc ttcaagggtg cccgctacta cgtgctggcc cgagggggac tg#caagtgga   1380gccctactac ccccgaagtc tgcaggactg gggaggcatc cctgaggagg tc#agcggcgc   1440cctgccgagg cccgatggct ccatcatctt cttccgagat gaccgctact gg#cgcctcga   1500ccaggccaaa ctgcaggcaa ccacctcggg ccgctgggcc accgagctgc cc#tggatggg   1560 ctgctggcat gccaactcgg ggagcgccct gttctga      #                   #    1597 <210> SEQ ID NO 4 <211> LENGTH: 513<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4Met Lys Arg Leu Leu Leu Leu Cys Leu Phe Ph #e Ile Thr Phe Ser Ser1               5    #                10   #                15Ala Phe Pro Leu Val Arg Met Thr Glu Asn Gl #u Glu Asn Met Gln Leu            20       #            25       #            30Ala Gln Ala Tyr Leu Asn Gln Phe Tyr Ser Le #u Glu Ile Glu Gly Asn        35           #        40           #        45His Leu Val Gln Ser Lys Asn Arg Ser Leu Il #e Asp Asp Lys Ile Arg    50               #    55               #    60Glu Met Gln Ala Phe Phe Gly Leu Thr Val Th #r Gly Lys Leu Asp Ser65                   #70                   #75                   #80Asn Thr Leu Glu Ile Met Lys Thr Pro Arg Cy #s Gly Val Pro Asp Val                85   #                90   #                95Gly Gln Tyr Gly Tyr Thr Leu Pro Gly Trp Ar #g Lys Tyr Asn Leu Thr            100       #           105       #           110Tyr Arg Ile Ile Asn Tyr Thr Pro Asp Met Al #a Arg Ala Ala Val Asp        115           #       120           #       125Glu Ala Ile Gln Glu Gly Leu Glu Val Trp Se #r Lys Val Thr Pro Leu    130               #   135               #   140Lys Phe Thr Lys Ile Ser Lys Gly Ile Ala As #p Ile Met Ile Ala Phe145                 1 #50                 1 #55                 1 #60Arg Thr Arg Val His Gly Arg Cys Pro Arg Ty #r Phe Asp Gly Pro Leu                165   #               170   #               175Gly Val Leu Gly His Ala Phe Pro Pro Gly Pr #o Gly Leu Gly Gly Asp            180       #           185       #           190Thr His Phe Asp Glu Asp Glu Asn Trp Thr Ly #s Asp Gly Ala Gly Phe        195           #       200           #       205Asn Leu Phe Leu Val Ala Ala His Glu Phe Gl #y His Ala Leu Gly Leu    210               #   215               #   220Ser His Ser Asn Asp Gln Thr Ala Leu Met Ph #e Pro Asn Tyr Val Ser225                 2 #30                 2 #35                 2 #40Leu Asp Pro Arg Lys Tyr Pro Leu Ser Gln As #p Asp Ile Asn Gly Ile                245   #               250   #               255Gln Ser Ile Tyr Gly Gly Leu Pro Lys Val Pr #o Ala Lys Pro Lys Glu            260       #           265       #           270Pro Thr Ile Pro His Ala Cys Asp Pro Asp Le #u Thr Phe Asp Ala Ile        275           #       280           #       285Thr Thr Phe Arg Arg Glu Val Met Phe Phe Ly #s Gly Arg His Leu Trp    290               #   295               #   300Arg Ile Tyr Tyr Asp Ile Thr Asp Val Glu Ph #e Glu Leu Ile Ala Ser305                 3 #10                 3 #15                 3 #20Phe Trp Pro Ser Leu Pro Ala Asp Leu Gln Al #a Ala Tyr Glu Asn Pro                325   #               330   #               335Arg Asp Lys Ile Leu Val Phe Lys Asp Glu As #n Phe Trp Met Ile Arg            340       #           345       #           350Gly Tyr Ala Val Leu Pro Asp Tyr Pro Lys Se #r Ile His Thr Leu Gly        355           #       360           #       365Phe Pro Gly Arg Val Lys Lys Ile Asp Ala Al #a Val Cys Asp Lys Thr    370               #   375               #   380Thr Arg Lys Thr Tyr Phe Phe Val Gly Ile Tr #p Cys Trp Arg Phe Asp385                 3 #90                 3 #95                 4 #00Glu Met Thr Gln Thr Met Asp Lys Gly Phe Pr #o Gln Arg Val Val Lys                405   #               410   #               415His Phe Pro Gly Ile Ser Ile Arg Val Asp Al #a Ala Phe Gln Tyr Lys            420       #           425       #           430Gly Phe Phe Phe Phe Ser Arg Gly Ser Lys Gl #n Phe Glu Tyr Asn Ile        435           #       440           #       445Lys Thr Lys Asn Ile Thr Arg Ile Met Arg Th #r Asn Thr Trp Phe Gln    450               #   455               #   460Cys Lys Glu Pro Lys Asn Ser Ser Phe Gly Ph #e Asp Ile Asn Lys Glu465                 4 #70                 4 #75                 4 #80Lys Ala His Ser Gly Gly Ile Lys Ile Leu Ty #r His Lys Ser Leu Ser                485   #               490   #               495Leu Phe Ile Phe Gly Ile Val His Leu Leu Ly #s Asn Thr Ser Ile Tyr            500       #           505       #           510 Gln<210> SEQ ID NO 5 <211> LENGTH: 259 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 5Met Gln Leu Val Ile Leu Arg Val Thr Ile Ph #e Leu Pro Trp Cys Phe1               5    #                10   #                15Ala Val Pro Val Pro Pro Ala Ala Asp His Ly #s Gly Trp Asp Phe Val            20       #            25       #            30Glu Gly Tyr Phe His Gln Phe Phe Leu Thr Ly #s Lys Glu Ser Pro Leu        35           #        40           #        45Leu Thr Gln Glu Thr Gln Thr Gln Leu Leu Gl #n Gln Phe His Arg Asn    50               #    55               #    60Gly Thr Asp Leu Leu Asp Met Gln Met His Al #a Leu Leu His Gln Pro65                   #70                   #75                   #80His Cys Gly Val Pro Asp Gly Ser Asp Thr Se #r Ile Ser Pro Gly Arg                85   #                90   #                95Cys Lys Trp Asn Lys His Thr Leu Thr Tyr Ar #g Ile Ile Asn Tyr Pro            100       #           105       #           110His Asp Met Lys Pro Ser Ala Val Lys Asp Se #r Ile Tyr Asn Ala Val        115           #       120           #       125Ser Ile Trp Ser Asn Val Thr Pro Leu Ile Ph #e Gln Gln Val Gln Asn    130               #   135               #   140Gly Asp Ala Asp Ile Lys Val Ser Phe Trp Gl #n Trp Ala His Glu Asp145                 1 #50                 1 #55                 1 #60Gly Trp Pro Phe Asp Gly Pro Gly Gly Ile Le #u Gly His Ala Phe Leu                165   #               170   #               175Pro Asn Ser Gly Asn Pro Gly Val Val His Ph #e Asp Lys Asn Glu His            180       #           185       #           190Trp Ser Ala Ser Asp Thr Gly Tyr Asn Leu Ph #e Leu Val Ala Thr His        195           #       200           #       205Glu Ile Gly His Ser Leu Gly Leu Gln His Se #r Gly Asn Gln Ser Ser    210               #   215               #   220Ile Met Tyr Pro Thr Tyr Trp Tyr His Asp Pr #o Arg Thr Phe Gln Leu225                 2 #30                 2 #35                 2 #40Ser Ala Asp Asp Ile Gln Arg Ile Gln His Le #u Tyr Gly Glu Lys Cys                245   #               250   #               255Ser Ser Asp <210> SEQ ID NO 6 <211> LENGTH: 520 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 6Met Val Ala Arg Val Gly Leu Leu Leu Arg Al #a Leu Gln Leu Leu Leu1               5    #                10   #                15Trp Gly His Leu Asp Ala Gln Pro Ala Glu Ar #g Gly Gly Gln Glu Leu            20       #            25       #            30Arg Lys Glu Ala Glu Ala Phe Leu Glu Lys Ty #r Gly Tyr Leu Asn Glu        35           #        40           #        45Gln Val Pro Lys Ala Pro Thr Ser Thr Arg Ph #e Ser Asp Ala Ile Arg    50               #    55               #    60Ala Phe Gln Trp Val Ser Gln Leu Pro Val Se #r Gly Val Leu Asp Arg65                   #70                   #75                   #80Ala Thr Leu Arg Gln Met Thr Arg Pro Arg Cy #s Gly Val Thr Asp Thr                85   #                90   #                95Asn Ser Tyr Ala Ala Trp Ala Glu Arg Ile Se #r Asp Leu Phe Ala Arg            100       #           105       #           110His Arg Thr Lys Met Arg Arg Lys Lys Arg Ph #e Ala Lys Gln Gly Asn        115           #       120           #       125Lys Trp Tyr Lys Gln His Leu Ser Tyr Arg Le #u Val Asn Trp Pro Glu    130               #   135               #   140His Leu Arg Ser Arg Gln Phe Gly Ala Pro Cy #s Ala Pro Pro Ser Ser145                 1 #50                 1 #55                 1 #60Cys Gly Ala Thr Ser Gln Arg Trp Ser Ser Gl #y Arg Pro Gln Pro Gln                165   #               170   #               175Ala Pro Leu Thr Ser Gly Ser Pro Ser Ser Ly #s Gly Thr Thr Thr Met            180       #           185       #           190Gly Trp Ala Met Pro Leu Met Ala Gln Gly Al #a Pro Trp Arg Thr Pro        195           #       200           #       205Phe Leu Pro Arg Arg Gly Glu Ala His Phe As #p Gln Asp Glu Arg Trp    210               #   215               #   220Ser Leu Ser Arg Arg Arg Gly Arg Asn Leu Ph #e Val Val Leu Ala His225                 2 #30                 2 #35                 2 #40Glu Ile Gly His Thr Leu Gly Leu Thr His Se #r Pro Ala Pro Arg Ala                245   #               250   #               255Leu Met Ala Pro Tyr Tyr Lys Arg Leu Gly Ar #g Asp Ala Leu Leu Ser            260       #           265       #           270Trp Asp Asp Val Leu Ala Val Gln Ser Leu Ty #r Gly Lys Pro Leu Gly        275           #       280           #       285Gly Ser Val Ala Val Gln Leu Pro Gly Lys Le #u Phe Thr Asp Phe Glu    290               #   295               #   300Thr Trp Asp Ser Tyr Ser Pro Gln Gly Arg Ar #g Pro Glu Thr Gln Gly305                 3 #10                 3 #15                 3 #20Pro Lys Tyr Cys His Ser Ser Phe Asp Ala Il #e Thr Val Asp Arg Gln                325   #               330   #               335Gln Gln Leu Tyr Ile Phe Lys Gly Ser His Ph #e Trp Glu Val Ala Ala            340       #           345       #           350Asp Gly Asn Val Ser Glu Pro Arg Pro Leu Gl #n Glu Arg Trp Val Gly        355           #       360           #       365Leu Pro Pro Asn Ile Glu Ala Ala Ala Val Se #r Leu Asn Asp Gly Asp    370               #   375               #   380Phe Tyr Phe Phe Lys Gly Gly Arg Cys Trp Ar #g Phe Arg Gly Pro Lys385                 3 #90                 3 #95                 4 #00Pro Val Trp Gly Leu Pro Gln Leu Cys Arg Al #a Gly Gly Leu Pro Arg                405   #               410   #               415His Pro Asp Ala Ala Leu Phe Phe Pro Pro Le #u Arg Arg Leu Ile Leu            420       #           425       #           430Phe Lys Gly Ala Arg Tyr Tyr Val Leu Ala Ar #g Gly Gly Leu Gln Val        435           #       440           #       445Glu Pro Tyr Tyr Pro Arg Ser Leu Gln Asp Tr #p Gly Gly Ile Pro Glu    450               #   455               #   460Glu Val Ser Gly Ala Leu Pro Arg Pro Asp Gl #y Ser Ile Ile Phe Phe465                 4 #70                 4 #75                 4 #80Arg Asp Asp Arg Tyr Trp Arg Leu Asp Gln Al #a Lys Leu Gln Ala Thr                485   #               490   #               495Thr Ser Gly Arg Trp Ala Thr Glu Leu Pro Tr #p Met Gly Cys Trp His            500       #           505       #           510Ala Asn Ser Gly Ser Ala Leu Phe         515           #       520<210> SEQ ID NO 7 <211> LENGTH: 999 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION:<301> AUTHORS: Park, H.I., Ni, J., Gerkema, F.E #., Liu, D., Belozero      Sang., Q.X. <302> TITLE: Identification and characterization of #human       endometalloproteinase-26) from endometrial # tumor<303> JOURNAL: J. Biol. Chem. <304> VOLUME: 275 <305> ISSUE: 27<306> PAGES: 20540-20544 <307> DATE: 2000-03-23<308> DATABASE ACCESSION NUMBER: GenBankAF248646<309> DATABASE ENTRY DATE: 2000-03-23 <300> PUBLICATION INFORMATION:<308> DATABASE ACCESSION NUMBER: GenbankAF248646<309> DATABASE ENTRY DATE: 2000-03-23 <400> SEQUENCE: 7ggcacgagca tgcagctcgt catcttaaga gttactatct tcttgccctg gt#gtttcgcc     60gttccagtgc cccctgctgc agaccataaa ggatgggact ttgttgaggg ct#atttccat    120caatttttcc tgaccgagaa ggagtcgcca ctccttaccc aggagacaca aa#cacagctc    180ctgcaacaat tccatcggaa tgggacagac ctacttgaca tgcagatgca tg#ctctgcta    240caccagcccc actgtggggt gcctgatggg tccgacacct ccatctcgcc ag#gaagatgc    300aagtggaata agcacactct aacttacagg attatcaatt acccacatga ta#tgaagcca    360tccgcagtga aagacagtat atataatgca gtttccatct ggagcaatgt ga#cccctttg    420atattccagc aagtgcagaa tggagatgca gacatcaagg tttctttctg gc#agtgggcc    480catgaagatg gttggccctt tgatgggcca ggtggtatct taggccatgc ct#ttttacca    540aattctggaa atcctggagt tgtccatttt gacaagaatg aacactggtc ag#cttcagac    600actggatata atctgttcct ggttgcaact catgagattg ggcattcttt gg#gcctgcag    660cactctggga atcagagctc cataatgtac cccacttact ggtatcacga cc#ctagaacc    720ttccagctca gtgccgatga tatccaaagg atccagcatt tgtatggaga aa#aatgttca    780tctgacatac cttaatgtta gcacagagga cttattcaac ctgtctttca gg#gagtttat    840tggaggatca aagaactgaa agcactagag cagccttggg gactgctagg at#gaagccct    900aaagaatgca acctagtcag gttagctgaa ccgacactca aaacgctact ga#gtcacaat    960 aaagattgtt ttaaagagta aaaaaaaaaa aaaaaaaaa      #                   #   999 <210> SEQ ID NO 8 <211> LENGTH: 261<212> TYPE: PRT <213> ORGANISM: Homo sapiens<300> PUBLICATION INFORMATION:<301> AUTHORS: Park, H.I., Ni, J., Gerkema, F.E#., Liu, D., Belozero Sang,       Q.X.<302> TITLE: Identification and characterization of  #human      endometalloproteinase-26) from endometrial # tumor<303> JOURNAL: J. Biol. Cehm. <304> VOLUME: 275 <305> ISSUE: 27<306> PAGES: 20540-205444 <307> DATE: 2000-03-23<308> DATABASE ACCESSION NUMBER: GenBankAF248626<309> DATABASE ENTRY DATE: 2001-03-23 <300> PUBLICATION INFORMATION:<308> DATABASE ACCESSION NUMBER: GenbankAF248646<309> DATABASE ENTRY DATE: 2000-03-23 <400> SEQUENCE: 8Met Gln Leu Val Ile Leu Arg Val Thr Ile Ph #e Leu Pro Trp Cys Phe1               5    #                10   #                15Ala Val Pro Val Pro Pro Ala Ala Asp His Ly #s Gly Trp Asp Phe Val            20       #            25       #            30Glu Gly Tyr Phe His Gln Phe Phe Leu Thr Gl #u Lys Glu Ser Pro Leu        35           #        40           #        45Leu Thr Gln Glu Thr Gln Thr Gln Leu Leu Gl #n Gln Phe His Arg Asn    50               #    55               #    60Gly Thr Asp Leu Leu Asp Met Gln Met His Al #a Leu Leu His Gln Pro65                   #70                   #75                   #80His Cys Gly Val Pro Asp Gly Ser Asp Thr Se #r Ile Ser Pro Gly Arg                85   #                90   #                95Cys Lys Trp Asn Lys His Thr Leu Thr Tyr Ar #g Ile Ile Asn Tyr Pro            100       #           105       #           110His Asp Met Lys Pro Ser Ala Val Lys Asp Se #r Ile Tyr Asn Ala Val        115           #       120           #       125Ser Ile Trp Ser Asn Val Thr Pro Leu Ile Ph #e Gln Gln Val Gln Asn    130               #   135               #   140Gly Asp Ala Asp Ile Lys Val Ser Phe Trp Gl #n Trp Ala His Glu Asp145                 1 #50                 1 #55                 1 #60Gly Trp Pro Phe Asp Gly Pro Gly Gly Ile Le #u Gly His Ala Phe Leu                165   #               170   #               175Pro Asn Ser Gly Asn Pro Gly Val Val His Ph #e Asp Lys Asn Glu His            180       #           185       #           190Trp Ser Ala Ser Asp Thr Gly Tyr Asn Leu Ph #e Leu Val Ala Thr His        195           #       200           #       205Glu Ile Gly His Ser Leu Gly Leu Gln His Se #r Gly Asn Gln Ser Ser    210               #   215               #   220Ile Met Tyr Pro Thr Tyr Trp Tyr His Asp Pr #o Arg Thr Phe Gln Leu225                 2 #30                 2 #35                 2 #40Ser Ala Asp Asp Ile Gln Arg Ile Gln His Le #u Tyr Gly Glu Lys Cys                245   #               250   #               255Ser Ser Asp Ile Pro             260

What is claimed is:
 1. An isolated nucleic acid molecule comprising asequence that encodes a polypeptide comprising the sequence of SEQ IDNO:6, said polypeptide having matrix metalloproteinase (MMP) proteaseactivity, with the proviso that the nucleotide sequence is not SEQ IDNO:7.
 2. The isolated nucleic acid molecule of claim 1 comprising asequence with at least 95% sequence homology to the sequence of SEQ IDNO:3.
 3. The isolated nucleic acid molecule of claim 1 comprising thesequence of SEQ ID NO:3.
 4. The isolated nucleic acid molecule of claim1 wherein said nucleic acid molecule is DNA.
 5. The isolated nucleicacid molecule of claim 1 wherein said nucleic acid molecule is RNA. 6.An isolated nucleic acid molecule comprising a sequence that encodes apolypeptide comprising a sequence at least 99% homologous to SEQ IDNO:6, said polypeptide having MMP protease activity, with the provisothat the nucleotide sequence is not SEQ ID NO:7 and wherein sequencehomology is determined using the Gap program with default settings. 7.The isolated nucleic acid molecule of claim 6 comprising a sequence withat least 90% sequence homology to the sequence of SEQ ID NO:3.
 8. Anexpression vector comprising a nucleic acid molecule of any one ofclaims 1 to 3, 6, and
 7. 9. The expression vector of claim 8 whereinsaid nucleic acid molecule comprises the sequence of SEQ ID NO:3. 10.The expression vector of claim 8 wherein said vector is a plasmid. 11.The expression vector of claim 8 wherein said vector is a viralparticle.
 12. The expression vector of claim 11 wherein said vector isselected from the group consisting of adenoviruses, baculoviruses,parvoviruses, herpes viruses, poxviruses, adeno-associated viruses,Semliki Forest viruses, vaccinia viruses, and retroviruses.
 13. Theexpression vector of claim 8 wherein said nucleic acid molecule isoperably connected to a promoter selected from the group consisting ofsimian virus 40, mouse mammary tumor virus, long terminal repeat ofhuman immunodeficiency virus, maloney virus, cytomegalovirus immediateearly promoter, Epstein Barr virus, rous sarcoma virus, human actin,human myosin, human hemoglobin, human muscle creatine kinase, and humanmetallothionein.
 14. A host cell transformed with an expression vectorof claim
 8. 15. The transformed host cell of claim 14 wherein said cellis a bacterial cell.
 16. The transformed host cell of claim 15 whereinsaid bacterial cell is E. coli.
 17. The transformed host cell of claim14 wherein said cell is yeast.
 18. The transformed host cell of claim 17wherein said yeast is S cerevisiae.
 19. The transformed host cell ofclaim 14 wherein said cell is an insect cell.
 20. The transformed hostcell of claim 19 wherein said insect cell is S. frugiperda.
 21. Thetransformed host cell of claim 14 wherein said cell is a mammalian cell.22. The transformed host cell of claim 21 wherein said mammalian cell isselected from the group consisting of chinese hamster ovary cells, HeLacells, African green monkey kidney cells, human HEK-293 cells, andmurine 3T3 fibroblasts.
 23. A composition comprising a nucleic acidmolecule of any one of claims 1 to 3, 6, and 7 and an acceptable carrieror diluent.
 24. A composition comprising a recombinant expression vectorof claim 8 and an acceptable carrier or diluent.
 25. A method ofproducing a polypeptide that comprises the sequence of SEQ ID NO:6, saidpolypeptide having MMP protease activity, said method comprising thesteps of: a) introducing a recombinant expression vector of claim 9 intoa compatible host cell; b) growing said host cell under conditions forexpression of said polypeptide; and c) recovering said polypeptide. 26.The method of claim 25 wherein said host cell is lysed and saidpolypeptide is recovered from the lysate of said host cell.
 27. Themethod of claim 25 wherein said polypeptide is recovered by purifyingthe culture medium without lysing said host cell.